Title 40—Protection of Environment--Volume 1
PART 799—IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE TESTING REQUIREMENTS
§ 799.9355   TSCA 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 870.3550 (July 2000, final guidelines). This source is available at the address in paragraph (h) of this section.
(b) Purpose. (1) This guideline is designed to generate limited information concerning the effects of a test substance 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 comprehensive test standards in §§ 799.9370 and 799.9380.
(2) This screening test guideline can be used to provide initial information on possible effects on reproduction and/or development, either at an early stage of assessing the toxicological properties of chemicals, or on 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, as a dose range finding study for more extensive reproduction/developmental studies, or when otherwise considered relevant.
(3) 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 relatively small numbers of animals in the dose groups, the selectivity of the end points, and the short duration of the study, this method will not provide evidence for definite claims of no effects.
(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 of dose, its frequency and the duration of dosing.
Dose is the amount of test substance administered. Dose is expressed as weight (g, mg) 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 kilograms per day).
(d) Principle of the test. (1) The test substance is administered in graduated doses to several groups of males and females. Males should be dosed for a minimum of four weeks and up to and including the day before scheduled sacrifice (this includes a minimum of two weeks prior to mating, during the mating period and, approximately, two weeks post-mating). In view of the limited pre-mating dosing period in males, fertility may not be a particular sensitive indicator of testicular toxicity. Therefore, a detailed histological examination of the testes is essential. The combination of a pre-mating dosing period of two weeks and subsequent mating/fertility observations with an overall dosing period of at least four 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 two 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 four days after delivery, up to and including the day before scheduled sacrifice.
(3) Duration of study, following acclimatization, is dependent on the female performance and is approximately 54 days, (at least 14 days premating, (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 period 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 standard 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 characterized 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 20% of the mean weight of each sex.
(2) Housing and feeding conditions. (i) The temperature in the experimental animal room should be 22 °C (±3°). 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 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 randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimized. The animals must be uniquely identified and kept in their cages for at least five days prior to the start of the study to allow for acclimatization 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 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 vehicles other than water the toxic characteristics of the vehicle must be known. The stability of the test substance in the vehicle should be determined.
(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 8 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 F 1 offspring from conception to day 4 post-partum.
(2) Dosage. (i) Generally, at least three test groups and a control group should be used. Dose levels may be based on information from acute toxicity tests or on results from repeated dose studies. 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. The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels should be selected in order to demonstrate any dose response relationships and no adverse effects 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 10) between dosages.
(3) Limit test. If an oral study at one dose level of at least 1000 mg/kg body weight/day or, for dietary or drinking water administration, an equivalent percentage in the diet, or drinking water 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 oral 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 concentration.
(4) Administration of doses. (i) The animals must be dosed with the test substance daily for seven 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 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 at least 2 weeks prior to mating, after they have been acclimatized for at least five 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. 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) The experimental schedule is given in the following figure 1.
(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 two 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.
(7) Observations. (i) Throughout the test period, general clinical observations should be made at least once a day, and more frequently when signs of toxicity are observed. They should be made preferably at the same time(s) each day, considering the peak period of anticipated effects after dosing. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality, should be recorded. These records should include time of onset, degree and duration of toxicity signs.
(ii) 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.
(iii) Live pups should be counted and sexed and litters weighed within 24 hours of parturition (day 1) and on day 4 post-partum. In addition to the observations on parent animals, described by paragraph (f)(7) 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 individually 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 1) and day 4 post-partum.
(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 via drinking water.
(9) Pathology —(i) Gross necropsy. (A) At the time of sacrifice or death during the study, the adult animals should be examined macroscopically for any abnormalities or pathological changes. 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 male adult animals should be weighed.
(C) Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, should, at least, be carefully examined externally for gross abnormalities.
(D) The ovaries, testes, epididymides, accessory sex organs and all organs showing macroscopic lesions of all adult animals should be preserved. 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.
(ii) Histopathology. (A) Detailed histological examination should be performed on the ovaries, testes and epididymides of the animals of the highest dose group and the control group. The other preserved organs may be examined when necessary. Examinations should be extended to the animals of other dosage groups when changes are seen in the highest dose group.
(B) Detailed testicular histopathological examination (e.g., using Bouin's fixative, paraffin embedding and transverse sections of 4-5 ±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. 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.
(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. This evaluation must 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 testis and epididymus must be considered along with the fertility data, when assessing male reproductive effects.
(iii) 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. 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. Because of the small group size, the use of historic control data (e.g., for litter size), where available, may also be useful as an aid to the interpretation 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 concentrations, 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 million (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) Effects on reproduction, including information on mating/precoital interval, fertility, fecundity and gestation duration.
(F) 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.
(G) Body weight at termination and organ weight data for the parental animals.
(H) Necropsy data, including number of implantations and number of corpora lutea.
(I) Calculations of pre- and postimplantation loss.
(J) Detailed description of histopathological findings.
(K) Statistical treatment of results, where appropriate.
(vi) Discussion of results.
(vii) Conclusions.
(4) Interpretation of results. The study will provide evaluations of reproduction/developmental toxicity associated with administration of repeated doses. It could provide an indication of the need to conduct further investigations and provides guidance in the design of subsequent studies.
(h) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) OECD (1995). Reproduction/Developmental Toxicity Screening Test, OECD 421, OECD Guidelines for Testing of Chemicals.
(2) [Reserved]
§ 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 §§ 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 § 799.9305 of this part and reproductive/developmental toxicity as described in § 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.
(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 ±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 °C (±3°). 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 F 1 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 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), 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.
(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), 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 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.
(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 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 ±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 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 at the addresses in § 700.17(b)(1) and (2) of this chapter.
(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., 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 Values and Effects of Amitraz. Toxicology and Applied Pharmacology, 108:267-283.
(6) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Method for the Routine Assessment of Fore- and Hindlimb Grip Strength of Rats and Mice. Neurobehavorial Toxicology, 1:233-236.
(7) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W., Tilson H.A., MacPhail R.C. (1991). Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicology and Teratology 13:599-609.
§ 799.9370   TSCA prenatal developmental toxicity.
(a) Scope This section is intended to meet the testing requirements under section 4 of TSCA. This guideline for developmental toxicity testing is designed to provide general information concerning the effects of exposure on the pregnant test animal and on the developing organism; this may include death, structural abnormalities, or altered growth and an assessment of maternal effects. For information on testing for functional deficiencies and other postnatal effects, the guidelines for the two-generation reproductive toxicity study and the developmental neurotoxicity study should be consulted.
(b) Source. The source material used in developing this TSCA test guideline is the OPPTS harmonized test guideline 870.3700 (February 1996 Public Draft). This source is available at the address in paragraph (h) of this section.
(c) Good laboratory practice standards. The study shall be conducted in compliance with 40 CFR Part 792—Good Laboratory Practice Standards.
(d) Principle of the test method. The test substance is administered to pregnant animals at least from implantation to one day prior to the expected day of parturition. Shortly before the expected date of delivery, the pregnant females are terminated, the uterine contents are examined, and the fetuses are processed for visceral and skeletal evaluation.
(e) Test procedures —(1) Animal selection —(i) Species and strain. It is recommended that testing be performed in the most relevant species, and that laboratory species and strains which are commonly used in prenatal developmental toxicity testing be employed. The preferred rodent species is the rat and the preferred non-rodent species is the rabbit.
(ii) Age. Young adult animals shall be used.
(iii) Sex. Nulliparous female animals shall be used at each dose level. Animals should be mated with males of the same species and strain, avoiding the mating of siblings, if parentage is known. Day 0 in the test is the day on which a vaginal plug and/or sperm are observed in the rodent or that insemination is performed or observed in the rabbit.
(iv) Number of animals. Each test and control group shall contain a sufficient number of animals to yield approximately 20 animals with implantation sites at necropsy.
(2) Administration of test and control substances —(i) Dose levels and dose selection. (A) At least three-dose levels and a concurrent control shall be used. Healthy animals shall be randomly assigned to the control and treatment groups, in a manner which results in comparable mean body weight values among all groups. The dose levels should be spaced to produce a gradation of toxic effects. Unless limited by the physical/chemical nature or biological properties of the test substance, the highest dose shall be chosen with the aim to induce some developmental and/or maternal toxicity but not death or severe suffering. In the case of maternal mortality, this should not be more than approximately 10%. The intermediate dose levels should produce minimal observable toxic effects. The lowest dose level should not produce any evidence of either maternal or developmental toxicity (i.e., the no-observed-adverse-effect level, NOAEL) or should be at or near the limit of detection for the most sensitive endpoint. Two- or four-fold intervals are frequently optimal for spacing the dose levels, and the addition of a fourth test group is often preferable to using very large intervals (e.g., more than a factor of 10) between dosages.
(B) It is desirable that additional information on metabolism and pharmacokinetics of the test substance be available to demonstrate the adequacy of the dosing regimen. This information should be available prior to testing.
(C) The highest dose tested need not exceed 1,000 mg/kg/day by oral or dermal administration, or 2 mg/L (or the maximum attainable concentration) by inhalation, unless potential human exposure data indicate the need for higher doses. If a test performed at the limit dose level, using the procedures described for this study, produces no observable toxicity and if an effect would not be expected based upon data from structurally related compounds, then a full study using three-dose levels may not be considered necessary.
(ii) Control group. (A) A concurrent control group shall be used. This group shall be a sham-treated control group or a vehicle-control group if a vehicle is used in administering the test substance.
(B) The vehicle control group should receive the vehicle in the highest volume used.
(C) If a vehicle or other additive is used to facilitate dosing, consideration should be given to the following characteristics: Effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals.
(iii) Route of administration. (A) The test substance or vehicle is usually administered orally by intubation.
(B) If another route of administration is used, for example, when the route of administration is based upon the principal route of potential human exposure, the tester shall provide justification and reasoning for its selection, and appropriate modifications may be necessary. Care should be taken to minimize stress on the maternal animals. For materials administered by inhalation, whole-body exposure is preferable to nose-only exposure due to the stress of restraint required for nose-only exposure.
(C) The test substance shall be administered at approximately the same time each day.
(D) When administered by gavage or dermal application, the dose to each animal shall be based on the most recent individual body weight determination.
(iv) Dosing schedule. At minimum, the test substance shall be administered daily from implantation to the day before cesarean section on the day prior to the expected day of parturition. Alternatively, if preliminary studies do not indicate a high potential for preimplantation loss, treatment may be extended to include the entire period of gestation, from fertilization to approximately 1 day prior to the expected day of termination.
(f) Observation of animals —(1) Maternal. (i) Each animal shall be observed at least once daily, considering the peak period of anticipated effects after dosing. Mortality, moribundity, pertinent behavioral changes, and all signs of overt toxicity shall be recorded at this cageside observation. In addition, thorough physical examinations shall be conducted at the same time maternal body weights are recorded.
(ii) Animals shall be weighed on day 0, at termination, and at least at 3-day intervals during the dosing period.
(iii) Food consumption shall be recorded on at least 3-day intervals, preferably on days when body weights are recorded.
(iv) (A) Females shall be terminated immediately prior to the expected day of delivery.
(B) Females showing signs of abortion or premature delivery prior to scheduled termination shall be killed and subjected to a thorough macroscopic examination.
(v) At the time of termination or death during the study, the dam shall be examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Evaluation of the dams during cesarean section and subsequent fetal analyses should be conducted without knowledge of treatment group in order to minimize bias.
(vi) (A) Immediately after termination or as soon as possible after death, the uteri shall be removed and the pregnancy status of the animals ascertained. Uteri that appear nongravid shall be further examined (e.g. by ammonium sulfide staining) to confirm the nonpregnant status.
(B) Each gravid uterus (with cervix) shall be weighed. Gravid uterine weights should not be obtained from dead animals if autolysis or decomposition has occurred.
(C) The number of corpora lutea shall be determined for pregnant animals.
(D) The uterine contents shall be examined for embryonic or fetal deaths and the number of viable fetuses. The degree of resorption shall be described in order to help estimate the relative time of death of the conceptus.
(2) Fetal. (i) The sex and body weight of each fetus shall be determined.
(ii) Each fetus shall be examined for external anomalies.
(iii) Fetuses shall be examined for skeletal and soft tissue anomalies (e.g. variations and malformations or other categories of anomalies as defined by the performing laboratory).
(A) For rodents, approximately one-half of each litter shall be prepared by standard techniques and examined for skeletal alterations, preferably bone and cartilage. The remainder shall be prepared and examined for soft tissue anomalies, using appropriate serial sectioning or gross dissection techniques. It is also acceptable to examine all fetuses by careful dissection for soft tissue anomalies followed by an examination for skeletal anomalies.
(B) For rabbits, all fetuses shall be examined for both soft tissue and skeletal alterations. The bodies of these fetuses should be evaluated by careful dissection for soft-tissue anomalies, followed by preparation and examination for skeletal anomalies. An adequate evaluation of the internal structures of the head, including the eyes, brain, nasal passages, and tongue, should be conducted for at least half of the fetuses.
(g) Data and reporting —(1) Treatment of results. Data shall be reported individually and summarized in tabular form, showing for each test group the types of change and the number of dams, fetuses, and litters displaying each type of change.
(2) Evaluation of study results. The following shall be provided:
(i) Maternal and fetal test results, including an evaluation of the relationship, or lack thereof, between the exposure of the animals to the test substance and the incidence and severity of all findings.
(ii) Criteria used for categorizing fetal external, soft tissue, and skeletal anomalies.
(iii) When appropriate, historical control data to enhance interpretation of study results. Historical data (on litter incidence and fetal incidence within litter), when used, should be compiled, presented, and analyzed in an appropriate and relevant manner. In order to justify its use as an analytical tool, information such as the dates of study conduct, the strain and source of the animals, and the vehicle and route of administration should be included.
(iv) Statistical analysis of the study findings should include sufficient information on the method of analysis, so that an independent reviewer/statistician can reevaluate and reconstruct the analysis. In the evaluation of study data, the litter should be considered the basic unit of analysis.
(v) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered.
(3) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, the following specific information shall be reported. Both individual and summary data should be presented.
(i) Species and strain.
(ii) Maternal toxic response data by dose, including but not limited to:
(A) The number of animals at the start of the test, the number of animals surviving, the number pregnant, and the number aborting.
(B) Day of death during the study or whether animals survived to termination.
(C) Day of observation of each abnormal clinical sign and its subsequent course.
(D) Body weight and body weight change data, including body weight change adjusted for gravid uterine weight.
(E) Food consumption and, if applicable, water consumption data.
(F) Necropsy findings, including gravid uterine weight.
(iii) Developmental endpoints by dose for litters with implants, including:
(A) Corpora lutea counts.
(B) Implantation data, number and percent of live and dead fetuses, and resorptions (early and late).
(C) Pre- and postimplantation loss calculations.
(iv) Developmental endpoints by dose for litters with live fetuses, including:
(A) Number and percent of live offspring.
(B) Sex ratio.
(C) Fetal body weight data, preferably by sex and with sexes combined.
(D) External, soft tissue, and skeletal malformation and variation data. The total number and percent of fetuses and litters with any external, soft tissue, or skeletal alteration, as well as the types and incidences of individual anomalies, should be reported.
(v) The numbers used in calculating all percentages or indices.
(vi) Adequate statistical treatment of results.
(vii) A copy of the study protocol and any amendments should be included.
(h) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Aliverti, V.L. et al. The extent of fetal ossification as an index of delayed development in teratogenicity studies in the rat. Teratology. 20:237-242 (1979).
(2) Barrow, M.V. and W.J. Taylor. A rapid method for detecting malformations in rat fetuses. Journal of Morphology 127:291-306 (1969).
(3) Burdi, A.R. Toluidine blue-alizarin red S staining of cartilage and bone in whole-mount skeltons in vitro. Stain Technolology. 40:45-48 (1965).
(4) Edwards, J.A. Ed. Woolam,D.H.M. The external development of the rabbit and rat embryo. Vol. 3. Advances in Teratology (Academic, NY, 1968).
(5) Fritz, H. Prenatal ossification in rabbits as indicative of fetal maturity. Teratology. 11:313-320 (1974).
(6) Fritz, H. and Hess, R. Ossification of the rat and mouse skeleton in the perinatal period. Teratology. 3:331-338 (1970).
(7) Gibson, J.P. et al. Use of the rabbit in teratogenicity studies. Toxicology and Applied Pharmacology. 9:398-408 (1966).
(8) Inouye, M. Differential staining of cartilage and bone in fetal mouse skeleton by alcian blue and alizarin red S. Congenital Anomalies. 16(3):171-173 (1976).
(9) Igarashi, E. et al. Frequence of spontaneous axial skeletal variations detected by the double staining technique for ossified and cartilaginous skeleton in rat fetuses. Congenital Anomalies. 32:381-391 (1992).
(10) Kimmel, C.A. et al. Skeletal development following heat exposure in the rat. Teratology. 47:229-242 (1993).
(11) Kimmel, C.A. and Francis, E.Z. Proceedings of the workshop on the acceptability and interpretation of dermal developmental toxicity studies. Fundamental and Applied Toxicology. 14:386-398 (1990).
(12) Kimmel, C.A. and C. Trammell. A rapid procedure for routine double staining of cartilage and bone in fetal and adult animals. Stain Technology. 56:271-273 (1981).
(13) Kimmel, C.A. and Wilson, J.G. Skeletal deviation in rats: malformations or variations? Teratology. 8:309-316 (1973).
(14) Marr, M.C. et al. Comparison of single and double staining for evaluation of skeletal development: the effects of ethylene glycol (EG) in CD rats. Teratology. 37:476 (1988).
(15) Marr, M.C. et al. Developmental stages of the CD (Sprague-Dawley) rat skeleton after maternal exposure to ethylene glycol. Teratology. 46:169-181 (1992).
(16) McLeod, M.J. Differential staining of cartilage and bone in whole mouse fetuses by Alcian blue and alizarin red S. Teratology. 22:299-301 (1980).
(17) Monie, I.W. et al. Dissection procedures for rat fetuses permitting alizarin red staining of skeleton and histological study of viscera. Supplement to Teratology Workshop Manual. pp. 163-173 (1965).
(18) Organisation for Economic Co-operation and Development, No. 414: Teratogenicity, Guideline for Testing of Chemicals. [C(83)44 (Final)] (1983).
(19) Salewski (Koeln), V.E. Faerbermethode zum makroskopischen nachweis von implantations stellen am uterus der ratte. Naunyn-Schmeidebergs Archiv für Pharmakologie und Experimentelle Pathologie. 247:367 (1964).
(20) Spark, C. and Dawson,A.B. The order and time of appearance of centers of ossification in the fore and hind limbs of the albino rat, with special reference to the possible influence of the sex factor. American Journal of Anatomy. 41:411-445 (1928).
(21) Staples, R.E. Detection of visceral alterations in mammalian fetuses. Teratology. 9(3):A37-A38 (1974).
(22) Staples, R.E. and Schnell, V.L. Refinements in rapid clearing technique in the KOH—alizarin red S method for fetal bone. Stain Technology. 39:61-63 (1964).
(23) Strong, R.M. The order time and rate of ossification of the albino rat ( mus norvegicus albinus ) skeleton. American Journal of Anatomy. 36: 313-355 (1928).
(24) Stuckhardt, J.L. and Poppe, S.M. Fresh visceral examination of rat and rabbit fetuses used in teratogenicity testing. Teratogenesis, Carcinogenesis, and Mutagenesis. 4:181-188 (1984).
(25) Van Julsingha, E.B. and Bennett,C.G. Eds. Neubert, D., Merker, H.J., and Kwasigroch, T.E. A dissecting procedure for the detection of anomalies in the rabbit foetal head. Methods in Prenatal Toxicology (University of Chicago, Chicago, IL, 1977) pp. 126-144.
(26) Whitaker, J. and Dix, D.M. Double-staining for rat foetus skeletons in teratological studies. Laboratory Animals. 13:309-310 (1979).
(27) Wilson, J.G. Eds. Wilson, J.G. and Warkany, J. Embryological considerations in teratology. Teratology: Principles and Techniques (University of Chicago, Chicago, IL, 1965) pp. 251-277.
§ 799.9380   TSCA reproduction and fertility effects.
(a) Scope. This section is intended to meet the testing requirements under section 4 of the TSCA. This section is for two-generation reproduction testing and is designed to provide general information concerning the effects of a test substance on the integrity and performance of the male and female reproductive systems, including gonadal function, the estrous cycle, mating behavior, conception, gestation, parturition, lactation, and weaning, and on the growth and development of the offspring. The study may also provide information about the effects of the test substance on neonatal morbidity, mortality, target organs in the offspring, and preliminary data on prenatal and postnatal developmental toxicity and serve as a guide for subsequent tests. Additionally, since the study design includes in utero as well as postnatal exposure, this study provides the opportunity to examine the susceptibility of the immature/neonatal animal.
(b) Source. The source material used in developing this TSCA test guideline is the OPPTS harmonized test guideline 870.3800 (February 1996 Public Draft). This source is available at the address in paragraph (g) of this section.
(c) Good laboratory practice standards. The study shall be conducted in compliance with 40 CFR part 792—Good Laboratory Practice Standards.
(d) Principle of the test method. The test substance is administered to parental (P) animals prior to and during their mating, during the resultant pregnancies, and through the weaning of their F1 offspring. The substance is then administered to selected F1 offspring during their growth into adulthood, mating, and production of an F2 generation, until the F2 generation is weaned.
(e) Test procedures —(1) Animal selection —(i) Species and strain. The rat is the most commonly used species for testing. If another mammalian species is used, the tester shall provide justification/reasoning for its selection, and appropriate modifications will be necessary. Healthy parental 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. Strains of low fecundity shall not be used.
(ii) Age. Parental (P) animals shall be 5 to 9 weeks old at the start of dosing. The animals of all test groups should be of uniform weight, age, and parity as nearly as practicable, and should be representative of the species and strain under study.
(iii) Sex. (A) For an adequate assessment of fertility, both males and females shall be studied.
(B) The females shall be nulliparous and nonpregnant.
(iv) Number of animals. Each control group shall contain a sufficient number of mating pairs to yield approximately 20 pregnant females. Each test group shall contain a similar number of mating pairs.
(v) Identification of animals. Each animal shall be assigned a unique identification number. For the P generation, this should be done before dosing starts. For the F1 generation, this should be done for animals selected for mating; in addition, records indicating the litter of origin shall be maintained for all selected F1 animals.
(2) Administration of test and control substances —(i) Dose levels and dose selection. (A) At least three-dose levels and a concurrent control shall be used. Healthy animals should be randomly assigned to the control and treatment groups, in a manner which results in comparable mean body weight values among all groups. The dose levels should be spaced to produce a gradation of toxic effects. Unless limited by the physical/chemical nature or biological properties of the test substance, the highest dose should be chosen with the aim to induce some reproductive and/or systemic toxicity but not death or severe suffering. In the case of parental mortality, this should not be more than approximately 10%. The intermediate dose levels should produce minimal observable toxic effects. The lowest dose level should not produce any evidence of either systemic or reproductive toxicity (i.e., the no-observed-adverse-effect level, NOAEL) or should be at or near the limit of detection for the most sensitive endpoint. Two- or four-fold intervals are frequently optimal for spacing the dose levels, and the addition of a fourth test group is often preferable to using very large intervals (e.g., more than a factor of 10) between dosages.
(B) It is desirable that additional information on metabolism and pharmacokinetics of the test substance be available to demonstrate the adequacy of the dosing regimen. This information should be available prior to testing.
(C) The highest dose tested should not exceed 1,000 mg/kg/day (or 20,000 ppm in the diet), unless potential human exposure data indicate the need for higher doses. If a test performed at the limit dose level, using the procedures described for this study, produces no observable toxicity and if an effect would not be expected based upon data from structurally related compounds, then a full study using three dose levels may not be considered necessary.
(ii) Control group. (A) A concurrent control group shall be used. This group shall be an untreated or sham treated group or a vehicle-control group if a vehicle is used in administering the test substance.
(B) If a vehicle is used in administering the test substance, the control group shall receive the vehicle in the highest volume used.
(C) If a vehicle or other additive is used to facilitate dosing, consideration should be given to the following characteristics: Effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals.
(D) If a test substance is administered in the diet and causes reduced dietary intake or utilization, the use of a pair-fed control group may be considered necessary.
(iii) Route of administration. (A) The test substance is usually administered by the oral route (diet, drinking water, or gavage).
(B) If administered by gavage or dermal application, the dosage administered to each animal prior to mating and during gestation and lactation shall be based on the individual animal body weight and adjusted weekly at a minimum.
(C) If another route of administration is used, for example, when the route of administration is based upon the principal route of potential human exposure, the tester should provide justification and reasoning for its selection, and appropriate modifications may be necessary. Care should be taken to minimize stress on the maternal animals and their litters during gestation and lactation.
(D) All animals should be dosed by the same method during the appropriate experimental period.
(iv) Dosing schedule. (A) The animals should be dosed with the test substance on a 7-days-a-week basis.
(B) Daily dosing of the parental (P) males and females shall begin when they are 5 to 9 weeks old. Daily dosing of the F1 males and females shall begin at weaning. For both sexes (P and F1), dosing shall be continued for at least 10 weeks before the mating period.
(C) Daily dosing of the P and F1 males and females shall continue until termination.
(3) Mating procedure —(i) Parental. (A) For each mating, each female shall be placed with a single randomly selected male from the same dose level (1:1 mating) until evidence of copulation is observed or either 3 estrous periods or 2 weeks has elapsed. Animals should be separated as soon as possible after evidence of copulation is observed. If mating has not occurred after 2 weeks or 3 estrous periods, the animals should be separated without further opportunity for mating. Mating pairs should be clearly identified in the data.
(B) Vaginal smears shall be collected daily and examined for all females during mating, until evidence of copulation is observed.
(C) Each day, the females shall be examined for presence of sperm or vaginal plugs. Day 0 of pregnancy is defined as the day a vaginal plug or sperm are found.
(ii) F1 mating. For mating the F1 offspring, 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 F2 generation.
(iii) Second mating. In certain instances, such as poor reproductive performance in the controls, or in the event of treatment-related alterations in litter size, the adults may be remated to produce an F1b or F2b litter. If production of a second litter is deemed necessary in either generation, the dams should be remated approximately 1-2 weeks following weaning of the last F1a or F2a litter.
(iv) Special housing. After evidence of copulation, animals that are presumed to be pregnant shall be caged separately in delivery or maternity cages. Pregnant animals shall be provided with nesting materials when parturition is near.
(v) Standardization of litter sizes. (A) Animals should be allowed to litter normally and rear their offspring to weaning. Standardization of litter sizes is optional.
(B) If standardization is performed, the following procedure should be used. On day 4 after birth, the size of each litter may be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four males and four females per litter or five males and five females per litter. Selective elimination of pups, i.e. based upon body weight, is not appropriate. Whenever the number of male or female pups prevents having four (or five) of each sex per litter, partial adjustment (for example, five males and three females, or four males and six females) is acceptable. Adjustments are not appropriate for litters of eight pups or less.
(4) Observation of animals —(i) Parental. (A) Throughout the test period, each animal shall be observed at least once daily, considering the peak period of anticipated effects after dosing. Mortality, moribundity, pertinent behavioral changes, signs of difficult or prolonged parturition, and all signs of overt toxicity shall be recorded at this cageside examination. In addition, thorough physical examinations should be conducted weekly on each animal.
(B) Parental animals (P and F1) shall be weighed on the first day of dosing and weekly thereafter. Parental females (P and F1) should be weighed at a minimum on approximately gestation days 0, 7, 14, and 21, and during lactation on the same days as the weighing of litters.
(C) During the premating and gestation periods, food consumption shall be measured weekly at a minimum. Water consumption should be measured weekly at a minimum if the test substance is administered in the water.
(D) Estrous cycle length and pattern should be evaluated by vaginal smears for all P and F1 females during a minimum of 3 weeks prior to mating and throughout cohabitation; care should be taken to prevent the induction of pseudopregnancy.
(E) For all P and F1 males at termination, sperm from one testis and one epididymis shall be collected for enumeration of homogenization-resistant spermatids and cauda epididymal sperm reserves, respectively. In addition, sperm from the cauda epididymis (or vas deferens) should be collected for evaluation of sperm motility and sperm morphology.
( 1 ) The total number of homogenization-resistant testicular sperm and cauda epididymal sperm should be enumerated. The method described in the reference under paragraph (g)(8) of this section may be used. 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 mincing and/or homogenizing of the remaining cauda tissue. Enumeration in only control and high-dose P and F1 males may be performed unless treatment-related effects are observed; in that case, the lower dose groups should also be evaluated.
( 2 ) An evaluation of epididymal (or vas deferens) sperm motility should be performed. Sperm should be recovered while minimizing damage (the evaluation techniques as described in the reference under paragraph (g)(8) of this section may be used), and the percentage of progressively motile sperm should be determined either subjectively or objectively. For objective evaluations, an acceptable counting chamber of sufficient depth can be used to effectively combine the assessment of motility with sperm count and sperm morphology. When computer-assisted motion analysis is performed, the derivation of progressive motility relies on user-defined thresholds for average path velocity and straightness or linear index. If samples are videotaped, or images otherwise recorded, at the time of necropsy, subsequent analysis of only control and high-dose P and F1 males may be performed unless treatment-related effects are observed; in that case, the lower dose groups should also be evaluated. In the absence of a video or digital image, all samples in all treatment groups should be analyzed at necropsy.
( 3 ) A morphological evaluation of an epididymal (or vas deferens) sperm sample shall be performed. Sperm (at least 200 per sample) should be examined as fixed, wet preparations (the techniques for such examinations is described in the references under paragraphs (g)(4) and (g)(8) of this section may be used) and classified as either normal (both head and midpiece/tail appear normal) or abnormal. Examples of morphologic sperm abnormalities would include fusion, isolated heads, and misshapen heads and/or tails. Evaluation of only control and high-dose P and F1 males may be performed unless treatment-related effects are observed; in that case, the lower dose groups should also be evaluated.
(ii) Offspring. (A) Each litter should be examined as soon as possible after delivery (lactation day 0) to establish the number and sex of pups, stillbirths, live births, and the presence of gross anomalies. Pups found dead on day 0 should be examined for possible defects and cause of death.
(B) Live pups should be counted, sexed, and weighed individually at birth, or soon thereafter, at least on days 4, 7, 14, and 21 of lactation, at the time of vaginal patency or balanopreputial separation, and at termination.
(C) The age of vaginal opening and preputial separation should be determined for F1 weanlings selected for mating. If there is a treatment-related effect in F1 sex ratio or sexual maturation, anogenital distance should be measured on day 0 for all F2 pups.
(5) Termination schedule. (i) All P and F1 adult males and females should be terminated when they are no longer needed for assessment of reproductive effects.
(ii) F1 offspring not selected for mating and all F2 offspring should be terminated at comparable ages after weaning.
(6) Gross necropsy. (i) At the time of termination or death during the study, all parental animals (P and F1) and when litter size permits at least three pups per sex per litter from the unselected F1 weanlings and the F2 weanlings shall be examined macroscopically for any structural abnormalities or pathological changes. Special attention shall be paid to the organs of the reproductive system.
(ii) Dead pups or pups that are terminated in a moribund condition should be examined for possible defects and/or cause of death.
(iii) At the time of necropsy, a vaginal smear should be examined to determine the stage of the estrous cycle. The uteri of all cohabited females should be examined, in a manner which does not compromise histopathological evaluation, for the presence and number of implantation sites.
(7) Organ weights. (i) At the time of termination, the following organs of all P and F1 parental animals shall be weighed:
(A) Uterus (with oviducts and cervix), ovaries.
(B) Testes, epididymides (total weights for both and cauda weight for either one or both), seminal vesicles (with coagulating glands and their fluids), and prostate.
(C) Brain, pituitary, liver, kidneys, adrenal glands, spleen, and known target organs.
(ii) For F1 and F2 weanlings that are examined macroscopically, the following organs shall be weighed for one randomly selected pup per sex per litter.
(A) Brain.
(B) Spleen and thymus.
(8) Tissue preservation. The following organs and tissues, or representative samples thereof, shall be fixed and stored in a suitable medium for histopathological examination.
(i) For the parental (P and F1) animals:
(A) Vagina, uterus with oviducts, cervix, and ovaries.
(B) One testis (preserved in Bouins fixative or comparable preservative), one epididymis, seminal vesicles, prostate, and coagulating gland.
(C) Pituitary and adrenal glands.
(D) Target organs, when previously identified, from all P and F1 animals selected for mating.
(E) Grossly abnormal tissue.
(ii) For F1 and F2 weanlings selected for macroscopic examination: Grossly abnormal tissue and target organs, when known.
(9) Histopathology —(i) Parental animals. Full histopathology of the organs listed in paragraph (e)(8)(i) of this section shall be performed for ten randomly chosen high dose and control P and F1 animals per sex, for those animals that were selected for mating. Organs demonstrating treatment-related changes shall also be examined for the remainder of the high-dose and control animals and for all parental animals in the low- and mid-dose groups. Additionally, reproductive organs of the low- and mid-dose animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire, or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility, or morphology were affected, shall be subjected to histopathological evaluation. Besides gross lesions such as atrophy or tumors, testicular histopathological examination should be conducted in order to 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. Examination of the intact epididymis should include the caput, corpus, and cauda, which can be accomplished by evaluation of a longitudinal section, and should be conducted in order to identify such lesions as sperm granulomas, leukocytic infiltration (inflammation), aberrant cell types within the lumen, or the absence of clear cells in the cauda epididymal epithelium. The postlactational 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 be conducted for all F1 females; the number of animals, ovarian section selection, and section sample size should be statistically appropriate for the evaluation procedure used. Examination should include enumeration of the number of primordial follicles, which can be combined with small growing follicles (see paragraphs (g)(1) and (g)(2) of this section), for comparison of treated and control ovaries.
(ii) Weanling. For F1 and F2 weanlings, histopathological examination of treatment-related abnormalities noted in macroscopic examination should be considered, if such evaluation were deemed appropriate and would contribute to the interpretation of the study data.
(f) Data and reporting —(1) Treatment of results. Data shall be reported individually and summarized in tabular form, showing for each test group the types of change and the number of animals displaying each type of change.
(2) Evaluation of study results. (i) An evaluation of test results, including the statistical analysis, shall be provided. This should include an evaluation of the relationship, or lack thereof, between the exposure of the animals to the test substance and the incidence and severity of all abnormalities.
(ii) When appropriate, historical control data should be used to enhance interpretation of study results. Historical data, when used, should be compiled, presented, and analyzed in an appropriate and relevant manner. In order to justify its use as an analytical tool, information such as the dates of study conduct, the strain and source of the animals, and the vehicle and route of administration should be included.
(iii) Statistical analysis of the study findings should include sufficient information on the method of analysis, so that an independent reviewer/statistician can reevaluate and reconstruct the analysis.
(iv) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered.
(3) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, the following specific information shall be reported. Both individual and summary data should be presented.
(i) Species and strain.
(ii) Toxic response data by sex and dose, including indices of mating, fertility, gestation, birth, viability, and lactation; offspring sex ratio; precoital interval, including the number of days until mating and the number of estrous periods until mating; and duration of gestation calculated from day 0 of pregnancy. The report should provide the numbers used in calculating all indices.
(iii) Day (week) of death during the study or whether animals survived to termination; date (age) of litter termination.
(iv) Toxic or other effects on reproduction, offspring, or postnatal growth.
(v) Developmental milestone data (mean age of vaginal opening and preputial separation, and mean anogenital distance, when measured).
(vi) Number of P and F1 females cycling pattern and mean estrous cycle length.
(vii) Day (week) of observation of each abnormal sign and its subsequent course.
(viii) Body weight and body weight change data by sex for P, F1, and F2 animals.
(ix) Food (and water, if applicable) consumption, food efficiency (body weight gain per gram of food consumed), and test material consumption for P and F1 animals, except for the period of cohabitation.
(x) Total cauda epididymal sperm number, homogenization-resistant testis spermatid number, number and percent of progressively motile sperm, number and percent of morphologically normal sperm, and number and percent of sperm with each identified anomaly.
(xi) Stage of the estrous cycle at the time of termination for P and F1 parental females.
(xii) Necropsy findings.
(xiii) Implantation data and postimplantation loss calculations for P and F1 parental females.
(xiv) Absolute and adjusted organ weight data.
(xv) Detailed description of all histopathological findings.
(xvi) Adequate statistical treatment of results.
(xvii) A copy of the study protocol and any amendments should be included.
(g) References. For additional backgound information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2).
(1) Gray, L.E. et al. A dose-response analysis of methoxychlor-induced alterations of reproductive development and function in the rat. Fundamental and Applied Toxicology. 12:92-108 (1989).
(2) Heindel, J.J. et al. Ed. Hirshfield, A.N. Histological assessment of ovarian follicle number in mice as a screen of ovarian toxicity. Growth Factors and the Ovary (Plenum, NY, 1989) pp. 421-426.
(3) Korenbrot, C.C. et al. Preputial separation as an external sign of pubertal development in the male rat. Biology of Reproduction. 17:298-303 (1977).
(4) Linder, R.E. et al. Endpoints of spermatoxicity in the rat after short duration exposures to fourteen reproductive toxicants. Reproductive Toxicology. 6:491-505 (1992).
(5) Manson, J.M. and Kang, Y.J. Ed. Hayes, A.W. Test methods for assessing female reproductive and developmental toxicology. Principles and Methods of Toxicology (Raven, NY, 1989).
(6) Organisation for Economic Co-operation and Development, No. 416: Two Generation Reproduction Toxicity Study, Guidelines for Testing of Chemicals. [C(83)44 (Final)] (1983).
(7) Pederson, T. and Peters, H. Proposal for classification of oocytes and follicles in the mouse ovary. Journal of Reproduction and Fertility. 17:555-557 (1988).
(8) Seed, J., Chapin, R.E. E.D. Clegg, L.A. Dostal, R.H. Foote, M.E. Hurtt, G.R. Klinefelter, S.L. Makris, S.D. Perreault, S. Schrader, D. Seyler, R. Sprando, K.A. Treinen, D.N.R. Veeramachaneni, and Wise, L.D. Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and dog: a consensus report. Reproductive Toxicology. 10(3):237-244 (1996).
(9) Smith, B.J. et al. Comparison of random and serial sections in assessment of ovarian toxicity. Reproductive Toxicology. 5:379-383 (1991).
(10) Thomas, J.A. Eds. M.O. Amdur, J. Doull, and C.D. Klaassen. Toxic responses of the reproductive system. Casarett and Doull's Toxicology (Pergamon, NY, 1991).
(11) Working, P.K. and Hurtt, M. Computerized videomicrographic analysis of rat sperm motility. Journal of Andrology. 8:330-337 (1987).
(12) Zenick, H. et al. Ed. Hayes, A.W. Assessment of male reproductive toxicity: a risk assessment approach. Principles and Methods of Toxicology (Raven, NY, 1994).
§ 799.9410   TSCA chronic toxicity.
(a) Scope —(1) Applicability. This section is intended to meet the testing requirement 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 870.4100 (August 1998, final guidelines). This source is available at the address in paragraph (h) of this section
(b) Purpose. The objective of a chronic toxicity study is to determine the effects of a substance in a mammalian species following prolonged and repeated exposure. A chronic toxicity study should generate data from which to identify the majority of chronic effects and to define long-term dose-response relationships. The design and conduct of chronic toxicity tests should allow for the detection of general toxic effects, including neurological, physiological, biochemical, and hematological effects and exposure-related morphological (pathological) effects.
(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.
Chronic toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by the oral, dermal, or inhalation routes of exposure.
Cumulative toxicity is the adverse effects of repeated doses occurring as a result of prolonged action on, or increased concentration of, the administered test substance or its metabolites in susceptible tissue.
Dose in a chronic toxicity study is the amount of test substance administered daily via the oral, dermal or inhalation routes for a period of at least 12 months. Dose is expressed as weight of the test substance (grams, milligrams) per unit body weight of test animal (milligram per kilogram), or as weight of the test substance in parts per million (ppm) in food or drinking water per day. For inhalation exposure, dose is expressed as weight of the test substance per unit volume of air (milligrams per liter) or as parts per million per day. For dermal exposure, dose is expressed as weight of the test substance (grams, milligrams) per unit body weight of the test animal (milligrams per kilogram) or as weight of the substance per unit of surface area (milligrams per square centimeter) per day.
No-observed-effects level (NOEL) is the maximum dose used in a study which produces no adverse effects. The NOEL is usually expressed in terms of the weight of a test substance given daily per unit weight of test animal (milligrams per kilogram per day).
Target organ is any organ of a test animal showing evidence of an effect induced by a test substance.
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg body weight (expected human exposure may indicate the need for a higher dose level), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data of structurally related compounds, a full study using three dose levels might not be necessary.
(e) Test procedures —(1) Animal selection —(i) Species and strain. Testing should be performed with two mammalian species, one a rodent and the other a nonrodent. The rat is the preferred rodent species. Commonly used laboratory strains must be employed.
(ii) Age/weight. (A) Testing must be started with young healthy animals as soon as possible after weaning and acclimatization.
(B) Dosing of rodents should generally begin no later than 8 weeks of age.
(C) Dosing of non-rodents should begin between 4 and 6 months of age and in no case later than 9 months of age.
(D) At commencement of the study, the weight variation of animals used should be within 20% of the mean weight for each sex.
(E) Studies using prenatal or neonatal animals may be recommended under special conditions.
(iii) Sex. (A) Equal numbers of animals of each sex should be used at each dose level.
(B) Females should be nulliparous and nonpregnant.
(iv) Numbers. (A) For rodents, at least 40 animals (20 males and 20 females) and for nonrodents at least 8 animals (4 females and 4 males) should be used at each dose level and concurrent control group.
(B) If interim sacrifices are planned, the number should be increased by the number of animals scheduled to be sacrificed during the course of the study.
(C) The number of animals at the termination of the study must be adequate for a meaningful and valid statistical evaluation of chronic effects. The Agency must be notified if excessive early deaths or other problems are encountered that might compromise the integrity of the study.
(D) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.
(E) Each animal should be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides should be identified by reference to the unique numbers assigned.
(v) Husbandry. (A) Rodents may be group-caged by sex, but the number of animals per cage must not interfere with clear observation of each animal. The biological properties of the test substance or toxic effects (e.g., morbidity, excitability) may indicate a need for individual caging. Rodents should be housed individually in dermal studies and during exposure in inhalation studies. Caging should be appropriate to the nonrodent species.
(B) The temperature of the experimental animal rooms should be at 22 ±3 °C.
(C) The relative humidity of the experimental animal rooms should be 50 ±20%.
(D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark.
(E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Animals should be fed and watered ad libitum with food replaced at least weekly.
(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least 5 days is recommended.
(2) Control and test substances. (i) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed it should not elicit toxic effects itself nor substantially alter the chemical or toxicological properties of the test substance. It is recommended that wherever possible the use of an aqueous solution be the first choice, followed by consideration of solution in oil, and finally, solution in other vehicles.
(ii) One lot of the test substance should be used, if possible, throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound, and, if technically feasible, the names and quantities of contaminants and impurities.
(iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture.
(3) Control groups. A concurrent control group is required. This group should be an untreated or sham-treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required.
(4) Satellite group. A satellite group of 40 animals (20 animals per sex) for rodents and 8 animals (4 animals per sex) for nonrodents may be treated with the high-dose level for 12 months and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment of appropriate length, normally not less than 28 days. In addition, a control group of 40 animals (20 animals per sex) for rodents and 8 animals (4 animals per sex) for nonrodents should be added to the satellite study.
(5) Dose levels and dose selections. (i) In chronic toxicity tests, it is desirable to determine a dose-response relationship as well as a NOEL. Therefore, at least three dose levels with a control group and, where appropriate, a vehicle control (corresponding to the concentration of the vehicle at the highest exposure level) should be used. Dose levels should be spaced to produce a gradation of effects. A rationale must be provided for the doses selected.
(ii) The highest-dose level should elicit signs of toxicity without substantially altering the normal life span of the animal. The highest dose should be determined based on the findings from a 90-day study to ensure that the dose used is adequate to assess the chronic toxicity of the test substance. Thus, the selection of the highest dose to be tested is dependent upon changes observed in several toxicological parameters in subchronic studies. The highest dose tested need not exceed 1,000 mg/kg/day. If dermal application of the test substance produces severe skin irritation, then it may be necessary either to terminate the study and choose a lower high-dose level or to reduce the dose level. Gross criteria for defining severe irritation would include ulcers, fissures, exudate/crust(eschar), dead tissue, or anything leading to destruction of the functional integrity of the epidermis (e.g. caking, open sores, fissuring, eschar). Histological criteria for defining severe irritation would include follicular and interfollicular crust, microulcer, mild/moderate degeneration/necrosis, moderate/marked epidermal edema, marked dermal edema, and marked inflammation.
(iii) The intermediate dose levels should be spaced to produce a gradation of toxic effects.
(iv) The lowest-dose level should produce no evidence of toxicity.
(6) Administration of the test substance. The three main routes of administration are oral, dermal, and inhalation. The choice of the route of administration depends upon the physical and chemical characteristics of the test substance and the form typifying exposure in humans.
(i) Oral studies. Ideally, the animals should be dosed by gavage or with capsules on a 7-day per week basis for a period of at least 12 months. However, based primarily on practical considerations, dosing by gavage or capsules on a 5-day per week schedule is acceptable. If the test substance is administered via in the drinking water or mixed in the diet, exposure should be on a 7-day per week basis.
(ii) Dermal studies. (A) Preparation of animal skin. Shortly before testing, fur should be clipped from not less than 10% of the body surface area for application of the test substance. In order to dose approximately 10% of the body surface, the area starting at the scapulae (shoulders) to the wing of the ileum (hipbone) and half way down the flank on each side of the animal should be shaved. Shaving should be carried out approximately 24 hours before dosing. Repeated clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care should be taken to avoid abrading the skin which could alter its permeability.
(B) Preparation of test substance. Liquid test substances are generally used undiluted, except as indicated in paragraph (e)(5)(ii) of this section. Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, with a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of, and penetration of the skin by, the test substance should be taken into account. The volume of application should be kept constant, e.g., less than 100 µL for the mouse and less than 300 µL for the rat. Different concentrations of test solution should be prepared for different dose levels.
(C) Administration of test substance. The duration of exposure should be at least for 12 months. Ideally, the animals should be treated with test substance for at least 6 hours per day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day. The test substance should be applied uniformly over the treatment site. The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible. For rats, the test substance may be held in contact with the skin with a porous gauze dressing and nonirritating tape if necessary. The test site should be further covered in a suitable manner to retain the gauze dressing plus test substance and to ensure that the animals cannot ingest the test substance. The application site should not be covered when the mouse is the species of choice. The test substance may be wiped from the skin after the six-hour exposure period to prevent ingestion.
(iii) Inhalation studies. (A) The animals should be exposed to the test substance for 6 hours per day on a 7-day per week basis, for a period of at least 12 months. However, based primarily on practical considerations, exposure for 6 hours per day on a 5-day per week basis is acceptable.
(B) The animals should be tested in dynamic inhalation equipment designed to sustain a minimum air flow of 10 air changes per hour, an adequate oxygen content of at least 19%, and uniform conditions throughout the exposure chamber. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into surrounding areas. It is not normally necessary to measure chamber oxygen concentration if airflow is adequate.
(C) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. When a whole body chamber is used, individual housing must be used to minimize crowding of the test animals and maximize their exposure to the test substance. To ensure stability of a chamber atmosphere, the total volume occupied by the test animals should not exceed 5% of the volume of the test chamber. It is recommended, but not required, that nose-only or head-only exposure be used for aerosol studies in order to minimize oral exposures due to animals licking compound off their fur. The animals should be acclimated and heat stress minimized.
(D) The temperature at which the test is performed should be maintained at 22 ±2 °C. The relative humidity should be maintained between 40-60%, but in certain instances (e.g., use of water vehicle) this may not be practicable.
(E) The rate of air flow should be monitored continuously but recorded at least three times during the exposure.
(F) Temperature and humidity should be monitored continuously but should be recorded at least every 30 min.
(G) The actual concentrations of the test substance should be measured in the breathing zone. During the exposure period, the actual concentrations of the test substance should be held as constant as practicable, monitored continuously or intermittently depending on the method of analysis. Chamber concentration may be measured using gravimetric or analytical methods, as appropriate. If trial run measurements are reasonably consistent (±10% for liquid aerosol, gas, or vapor; ±20% for dry aerosol), then two measurements should be sufficient. If measurements are not consistent, three to four measurements should be taken. If there is some difficulty measuring chamber analytical concentration due to precipitation, nonhomogeneous mixtures, volatile components, or other factors, additional analysis of inert components may be necessary.
(H) During the development of the generating system, particle size analysis should be performed to establish the stability of aerosol concentrations with respect to particle size. The mass median aerodynamic diameter (MMAD) particle size range should be between 1-3 µm. The particle size of hygroscopic materials should be small enough when dry to assure that the size of the swollen particle will still be within the 1-3 µm range. Measurements of aerodynamic particle size in the animal's breathing zone should be measured during a trial run. If MMAD values for each exposure level are within 10% of each other, then two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, three to four measurements should be taken.
(I) Feed should be withheld during exposure. Water may also be withheld during exposure.
(7) Observation period. (i) Animals should be observed for a period of at least 12 months.
(ii) Animals in a satellite group (if used) scheduled for follow-up observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.
(8) Observation of animals. (i) Observations should be made at least twice each day for morbidity and mortality. Appropriate actions should be taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals). General clinical observations should be made at least once a day, preferably at the same time each day, taking into consideration the peak period of anticipated effects after dosing. The clinical condition of the animal should be recorded.
(ii) A careful clinical examination should be made at least once prior to the initiation of treatment (to allow for within subject comparisons) and once weekly during treatment in all animals. These observations should be made outside the home cage, preferably in a standard arena, and at similar times on each occasion. Effort should be made to ensure that variations in the observation conditions are minimal. Observations should be detailed and carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backwards) should be recorded.
(iii) Once, near the end of the first year of the exposure period and in any case not earlier than in month 11, assessment of motor activity, grip strength, and sensory reactivity to stimuli of different types (e.g., visual, auditory, and proprioceptive stimuli) should be conducted in rodents. Further details of the procedures that could be followed are described in the references listed under paragraphs (h)(2), (h)(7), (h)(8), and (h)(11) of this section.
(iv) Functional observations conducted towards the end of the study may be omitted when data on functional observations are available from other studies and the daily clinical observations did not reveal any functional deficits.
(v) Exceptionally, functional observations may be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with functional test performance.
(vi) Body weights should be recorded individually for all animals once prior to the administration of the test substance, once a week during the first 13 weeks of study and at least once every 4 weeks thereafter, unless signs of clinical toxicity suggest more frequent weighing to facilitate monitoring of health status.
(vii) Measurements of feed consumption should be determined weekly during the first 13 weeks of the study and at approximately monthly intervals thereafter unless health status or body weight changes dictate otherwise. Measurements of water consumption should be determined at the same intervals if the test substance is administered in the drinking water.
(viii) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. All survivors should be sacrificed at the end of the study period.
(9) Clinical pathology. Hematology, clinical chemistry, and urinalysis should be performed on 10 rats per sex per group, and on all nonrodents. In rodents, the parameters should be examined at approximately 6 month intervals during the conduct of the study and at termination. If possible, these collections should be from the same animals at each interval. In nonrodents, the parameters should be examined once or twice prior to initiation of treatment, at 6-month intervals during the conduct of the study, and at termination. If hematological and biochemical effects were seen in the subchronic study, testing should also be performed at 3 months. Overnight fasting of animals prior to blood sampling is recommended.
(i) Hematology. The recommended parameters are red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, white blood cell count, differential leukocyte count, platelet count, and a measure of clotting potential, such as prothrombin time or activated partial thromboplastin time.
(ii) Clinical chemistry. (A) Parameters which are considered appropriate to all studies are electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance and signs of clinical toxicity.
(B) The recommended clinical chemistry determinations are potassium, sodium, calcium (nonrodent), phosphorus (nonrodent), chloride (nonrodent), glucose, total cholesterol, urea nitrogen, creatinine, total protein, total bilirubin (nonrodent), and albumin. More than two hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of additional enzymes (of hepatic or other origin) and bile acids, may also be useful.
(C) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated.
(D) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases.
(iii) Urinalysis. Urinalysis for rodents should be performed at the end of the study using timed urine collection. Urinalysis for nonrodents should be performed prior to treatment, midway through treatment and at the end of the study using timed urine collection. Urinalysis determinations include: appearance, volume, osmolality or specific gravity, pH, protein, glucose, and blood/blood cells.
(10) Ophthalmological examination. Examinations should be made of all animals using an ophthalmoscope or equivalent device prior to the administration of the test substance and at termination of the study on 10 rats of each sex in the high-dose and control groups and preferably in all nonrodents, but at least the control and high-dose groups should be examined. If changes in eyes are detected, all animals should be examined.
(11) Gross necropsy. (i) All animals should be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
(ii) At least the liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, nonrodent thyroid (with parathyroid), spleen, brain, and heart should be weighed wet as soon as possible after dissection to avoid drying. The lungs should be weighed if the test substance is administered by the inhalation route.
(iii) The following organs and tissues, or representative samples thereof, should be preserved in a suitable medium for possible future histopathological examination:
(A) Digestive system—salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present).
(B) Nervous system—brain (multiple sections, including cerebrum, cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle), spinal cord (three levels, cervical, mid-thoracic and lumbar), eyes (retina, optic nerve).
(C) Glandular system—adrenals, parathyroid, thyroid.
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose.
(E) Cardiovascular/hematopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), spleen.
(F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
(G) Other—all gross lesions and masses, skin.
(iv) In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx, and paranasal sinuses should be examined and preserved. In dermal studies, skin from treated and adjacent control skin sites should be examined and preserved.
(v) Inflation of lungs and urinary bladder with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in inhalation studies is considered essential for appropriate and valid histopathological examination.
(vi) Information from clinical pathology and other in-life data should be considered before microscopic examination, since they may provide significant guidance to the pathologist.
(12) Histopathology. (i) The following histopathology should be performed:
(A) Full histopathology on the organs and tissues (listed under paragraph (e)(11)(iii) of this section) of all rodents and nonrodents in the control and high-dose groups, and all rodents and nonrodents that died or were sacrificed during the study. The examination should be extended to all animals in all dosage groups if treatment-related changes are observed in the high-dose group.
(B) All gross lesions in all animals.
(C) Target tissues in all animals.
(ii) If the results show substantial alteration of the animal's normal life span, or other effects that might compromise the significance of the data, the next lower levels should be examined fully as described in paragraph (e)(12)(i) of this section.
(iii) An attempt should be made to correlate gross observations with microscopic findings.
(iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming.
(f) Data and reporting —(1) Treatment of results. (i) Data should be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion.
(ii) When applicable, all observed results (quantitative and qualitative) should be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance criteria should be selected during the design of the study.
(2) Evaluation of study results. The findings of a chronic toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects as well as the necropsy and histopathological findings. The evaluation will include the relationship between the dose of the test substance and the presence, incidence, and severity of abnormalities (including behavioral and clinical abnormalities), gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects.
(3) Test report. In addition to the reporting requirements specified under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart J, the following specific information must be reported:
(i) Test substance characterization should include:
(A) Chemical identification.
(B) Lot or batch number.
(C) Physical properties.
(D) Purity/impurities.
(ii) Identification and composition of any vehicle used.
(iii) Test system should contain data on:
(A) Species and strain of animals used and rationale for selection if other than that recommended.
(B) Age including body weight data and sex.
(C) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods.
(D) Identification of animal diet.
(E) Acclimation period.
(iv) Test procedure should include the following data:
(A) Method of randomization used.
(B) Full description of experimental design and procedure.
(C) Dose regimen including levels, methods, and volume.
(v) Test results.
(A) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:
( 1 ) Number of animals exposed.
( 2 ) Number of animals showing signs of toxicity.
( 3 ) Number of animals dying.
(B) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals.
( 1 ) Time of death during the study or whether animals survived to termination.
( 2 ) Time of observation of each abnormal sign and its subsequent course.
( 3 ) Body weight data.
( 4 ) Feed and water (if collected) consumption data.
( 5 ) Achieved dose (mg/kg/day) as a time-weighted average if the test substance is administered in the diet or drinking water.
( 6 ) Results of ophthalmological examinations.
( 7 ) Results of hematological tests performed.
( 8 ) Results of clinical chemistry tests performed.
( 9 ) Urinalysis tests performed and results.
( 10 ) Results of observations made.
( 11 ) Necropsy findings, including absolute and relative (to body weight) organ weight data.
( 12 ) Detailed description of all histopathological findings.
( 13 ) Statistical treatment of results, where appropriate.
(vi) In addition, for inhalation studies the following should be reported:
(A) Test conditions. The following exposure conditions must be reported:
( 1 ) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulate and aerosols, method of conditioning air, treatment of exhaust air and the method of housing the animals in a test chamber.
( 2 ) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size should be described.
(B) Exposure data. These data should be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and should include:
( 1 ) Airflow rates through the inhalation equipment.
( 2 ) Temperature and humidity of air.
( 3 ) Actual (analytical or gravimetric) concentration in the breathing zone.
( 4 ) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
( 5 ) Particle size distribution, calculated MMAD, and geometric standard deviation.
( 6 ) Explanation as to why the desired chamber concentration and/or particle size could not be achieved (if applicable) and the efforts taken to comply with this aspect of the guidelines.
(g) Quality control. A system should be developed and maintained to assure and document adequate performance of laboratory staff and equipment. The study must be conducted in compliance with 40 CFR Part 792—Good Laboratory Practice Standards.
(h) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Benitz, K.F. Measurement of Chronic Toxicity. Methods of Toxicology. Ed. G.E. Paget. Blackwell, Oxford. pp. 82-131 (1970).
(2) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Leiter L.W., Tilson H.A., MacPhail, R.C. Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicol. Teratol. 13, 599-609. (1991)
(3) D'Aguanno, W. Drug Safety Evaluation-Pre-Clinical Considerations. Industrial Pharmacology: Neuroleptic. Vol. I, Ed. S. Fielding and H. Lal. Futura, Mt. Kisco, NY. pp. 317-332 (1974).
(4) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States. pp. 36-45 (1959, 3rd Printing 1975).
(5) Gad S.C. A Neuromuscular Screen for Use in Industrial Toxicology. Journal of Toxicology and Environmental Health. 9, 691-704. (1982)
(6) Goldenthal, E.I. and D'Aguanno, W. Evaluation of Drugs, Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The Association of Food and Drug Officials of the United States. pp. 60-67 (1959, 3rd Printing 1975).
(7) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. A Method for the Routine Assessment of Fore- and Hind-Limb Grip Strength of Rats and Mice. Neurobehav. Toxicol. 1, 233-236. (1979)
(8) Moser V.C., McDaniel K.M., Phillips P.M. Rat Strain and Stock Comparisons using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol. 108, 267-283 (1991)
(9) Organization for Economic Cooperation and Development. Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 452 Chronic Toxicity Studies, Paris (1981).
(10) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines. Journal of Environmental Pathology and Toxicology. 11:161-182 (1977).
(11) Tupper, D.E., Wallace R.B. Utility of the Neurologic Examination in Rats. Acta. Neurobiol. Exp. 40, 999-1003 (1980).
(12) Weingand K., Brown G., Hall R. et al. (1996). Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies. Fundam. and Appl. Toxicol. 29:198-201.
§ 799.9420   TSCA carcinogenicity.
(a) Scope. This section is intended to meet the testing requirements under section 4 of TSCA. The objective of a long-term carcinogenicity study is to observe test animals for a major portion of their life span for development of neoplastic lesions during or after exposure to various doses of a test substance by an appropriate route of administration.
(b) Source. The source material used in developing this TSCA test guideline is the OPPTS harmonized test guideline 870.4200 (June 1996 Public Draft). This source is available at the address in paragraph (g) of this section.
(c) Definitions. The following definitions apply to this section.
Carcinogenicity is the development of neoplastic lesions as a result of the repeated daily exposure of experimental animals to a chemical by the oral, dermal, or inhalation routes of exposure.
Cumulative toxicity is the adverse effects of repeated dose occurring as a result of prolonged action on, or increased concentration of, the administered test substance or its metabolites in susceptible tissues.
Dose in a carcinogenicity study is the amount of test substance administered via the oral, dermal or inhalation routes for a period of up to 24 months. Dose is expressed as weight of the test substance (grams, milligrams) per unit body weight of test animal (milligram per kilogram), or as weight of the test substance in parts per million (ppm) in food or drinking water. When exposed via inhalation, dose is expressed as weight of the test substance per unit volume of air (milligrams per liter) or as parts per million.
Target organ is any organ of a test animal showing evidence of an effect induced by a test substance.
(d) Test procedures —(1) Animal selection —(i) Species and strain. Testing shall be performed on two mammalian species. Rats and mice are the species of choice because of their relatively short life spans, limited cost of maintenance, widespread use in pharmacological and toxicological studies, susceptibility to tumor induction, and the availability of inbred or sufficiently characterized strains. Commonly used laboratory strains shall be used. If other mammalian species are used, the tester shall provide justification/reasoning for their selection.
(ii) Age/weight. (A) Testing shall be started with young healthy animals as soon as possible after weaning and acclimatization.
(B) Dosing should generally begin no later than 8 weeks of age.
(C) At commencement of the study, the weight variation of animals used shall not exceed ±20% of the mean weight for each sex.
(D) Studies using prenatal or neonatal animals may be recommended under special conditions.
(iii) Sex. (A) Equal numbers of animals of each sex shall be used at each dose level.
(B) Females shall be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 100 rodents (50 males and 50 females) shall be used at each dose level and concurrent control group.
(B) If interim sacrifices are planned, the number shall be increased by the number of animals scheduled to be sacrificed during the course of the study.
(C) For a meaningful and valid statistical evaluation of long term exposure and for a valid interpretation of negative results, the number of animals in any group should not fall below 50% at 15 months in mice and 18 months in rats. Survival in any group should not fall below 25% at 18 months in mice and 24 months in rats.
(D) The use of adequate randomization procedures for the proper allocation of animals to test and control groups is required to avoid bias.
(E) Each animal shall be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides shall be identified by reference to the unique numbers assigned.
(v) Husbandry. (A) Animals may be group-caged by sex, but the number of animals per cage must not interfere with clear observation of each animal. The biological properties of the test substance or toxic effects (e.g., morbidity, excitability) may indicate a need for individual caging. Animals should be housed individually in dermal studies and during exposure in inhalation studies.
(B) The temperature of the experimental animal rooms should be at 22 ±3 °C.
(C) The relative humidity of the experimental animal rooms should be 30 to 70%.
(D) Where lighting is artificial, the sequence should be 12 h light/12 h dark.
(E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure uniform distribution and adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Animals should be fed and watered ad libitum with food replaced at least weekly.
(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine.
(2) Control and test substances. (i) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed, it should not elicit toxic effects itself. It is recommended that wherever possible the use of an aqueous solution be considered first, followed by consideration of solution in oil, and finally solution in other vehicles.
(ii) One lot of the test substance should be used, if possible, throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound, and, if possible, the name and quantities of contaminants and impurities.
(iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture.
(3) Control groups. A concurrent control group (50 males and 50 females) is required. This group shall be untreated or if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known, both untreated and vehicle control groups are required.
(4) Dose levels and dose selection. (i) For risk assessment purposes, at least three dose levels shall be used, in addition to the concurrent control group. Dose levels should be spaced to produce a gradation of effects. A rationale for the doses selected must be provided.
(ii) The highest dose level should elicit signs of toxicity without substantially altering the normal life span due to effects other than tumors. The highest dose should be determined based on the findings from a 90-day study to ensure that the dose used is adequate to asses the carcinogenic potential of the test substance. Thus, the selection of the highest dose to be tested is dependent upon changes observed in several toxicological parameters in subchronic studies. The highest dose tested need not exceed 1,000 mg/kg/day.
(iii) The intermediate-dose level should be spaced to produce a gradation of toxic effects.
(iv) The lowest dose level should produce no evidence of toxicity.
(v) For skin carcinogenicity studies, when toxicity to the skin is a determining factor, the highest dose selected should not destroy the functional integrity of the skin, the intermediate dose should be a minimally irritating dose, and the low dose should be the highest nonirritating dose.
(vi) The criteria for selecting the dose levels for skin carcinogenicity studies, based on gross and histopathologic dermal lesions, are as follows:
(A) Gross criteria for reaching the high dose:
( 1 ) Erythema (moderate).
( 2 ) Scaling.
( 3 ) Edema (mild).
( 4 ) Alopecia.
( 5 ) Thickening.
(B) Histologic criteria for reaching the high dose:
( 1 ) Epidermal hyperplasia.
( 2 ) Epidermal hyperkeratosis.
( 3 ) Epidermal parakeratosis.
( 4 ) Adnexal atrophy/hyperplasia.
( 5 ) Fibrosis.
( 6 ) Spongiosis (minimal-mild).
( 7 ) Epidermal edema (minimal-mild).
( 8 ) Dermal edema (minimal-moderate).
( 9 ) Inflammation (moderate).
(C) Gross criteria for exceeding the high dose:
( 1 ) Ulcers, fissures.
( 2 ) Exudate/crust (eschar).
( 3 ) nonviable (dead) tissues.
( 4 ) Anything leading to destruction of the functional integrity of the epidermis (e.g., caking, fissuring, open sores, eschar).
(D) Histologic criteria for exceeding the high dose:
( 1 ) Crust (interfollicular and follicular).
( 2 ) Microulcer.
( 3 ) Degeneration/necrosis (mild to moderate).
( 4 ) Epidermal edema (moderate to marked).
( 5 ) Dermal edema (marked).
( 6 ) Inflammation (marked).
(5) Administration of the test substance. The three main routes of administration are oral, dermal, and inhalation. The choice of the route of administration depends upon the physical and chemical characteristics of the test substance and the form typifying exposure in humans.
(i) Oral studies. If the test substance is administered by gavage, the animals are dosed with the test substance on a 7-day per week basis for a period of at least 18 months for mice and hamsters and 24 months for rats. However, based primarily on practical considerations, dosing by gavage or via a capsule on a 5-day per week basis is acceptable. If the test substance is administered in the drinking water or mixed in the diet, then exposure should be on a 7-day per week basis.
(ii) Dermal studies. (A) The animals should be treated with the test substance for at least 6 h/day on a 7-day per week basis for a period of at least 18 months for mice and hamsters and 24 months for rats. However, based primarily on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day.
(B) Fur should be clipped weekly from the dorsal area of the trunk of the test animals. Care should be taken to avoid abrading the skin which could alter its permeability. A minimum of 24 hrs should be allowed for the skin to recover before the next dosing of the animal.
(C) Preparation of test substance. Liquid test substances are generally used undiluted, except as indicated in paragraph (e)(4)(vi) of this section. Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, with a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of, and penetration of the skin by, the test substance should be taken into account. The volume of application should be kept constant, e.g. less than 100 uL for the mouse and less than 300 uL for the rat. Different concentrations of test solution should be prepared for different dose levels.
(D) The test substance shall be applied uniformly over a shaved area which is approximately 10 percent of the total body surface area. In order to dose approximately 10 percent of the body surface, the area starting at the scapulae (shoulders) to the wing of the ileum (hipbone) and half way down the flank on each side of the animal should be shaved. With highly toxic substances, the surface area covered may be less, but as much of the area as possible should be covered with as thin and uniform a film as practical.
(iii) Inhalation studies. (A) The animals should be exposed to the test substance for 6 h/day on a 7-day per week basis, for a period of at least 18 months in mice and 24 months in rats. However, based primarily on practical considerations, exposure for 6 h/day on a 5-day per week basis is acceptable.
(B) The animals shall be tested in dynamic inhalation equipment designed to sustain a minimum air flow of 10 air changes per hr, an adequate oxygen content of at least 19%, and uniform conditions throughout the exposure chamber. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into surrounding areas.
(C) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. Where a whole body chamber is used to expose animals to an aerosol, individual housing must be used to minimize crowding of the test animals and maximize their exposure to the test substance. To ensure stability of a chamber atmosphere, the total volume occupied by the test animals shall not exceed 5% of the volume of the test chamber. It is recommended, but not required, that nose-only or head-only exposure be used for aerosol studies in order to minimize oral exposures due to animals licking compound off their fur. Heat stress to the animals should be minimized.
(D) The temperature at which the test is performed should be maintained at 22 ±2 °C. The relative humidity should be maintained between 40 to 60%, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable.
(E) The rate of air flow shall be monitored continuously but recorded at least three times during exposure.
(F) Temperature and humidity shall be monitored continuously but should be recorded at least every 30 minutes.
(G) The actual concentration of the test substance shall be measured in the breathing zone. During the exposure period, the actual concentrations of the test substance should be held as constant as practicable, monitored continuously or intermittently depending on the method of analysis. Chamber concentrations may be measured using gravimetric or analytical methods as appropriate. If trial run measurements are reasonably consistent (plus or minus 10 percent for liquid aerosol, gas, or vapor; plus or minus 20 percent for dry aerosol), the two measurements should be sufficient. If measurements are not consistent, then three to four measurements should be taken.
(H) During the development of the generating system, particle size analysis shall be performed to establish the stability of aerosol concentrations with respect to particle size. Measurement of aerodynamic particle size in the animals's breathing zone should be measured during a trial run. If median aerodynamic diameter (MMAD) values for each exposure level are within 10% of each other, then two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, three to four measurements should be taken. The MMAD particle size range should be between 1-3 µm. The particle size of hygroscopic materials should be small enough to allow pulmonary deposition once the particles swell in the moist environment of the respiratory tract.
(I) Feed shall be withheld during exposure. Water may also be withheld during exposure.
(6) Observation period. It is necessary that the duration of the carcinogenicity study comprise the majority of the normal life span of the strain of animals used. This time period shall not be less than 24 months for rats and 18 months for mice, and ordinarily not longer than 30 months for rats and 24 months for mice. For longer time periods, and where any other species are used, consultation with the Agency in regard to the duration of the study is advised.
(7) Observation of animals. (i) Observations shall be made at least once each day for morbidity and mortality. Appropriate actions should be taken to minimize loss of animals from the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals).
(ii) A careful clinical examination shall be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicitly defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards).
(iii) Body weights shall be recorded individually for all animals; once a week during the first 13 weeks of the study and at least once every 4 weeks, thereafter, unless signs of clinical toxicity suggest more frequent weighing to facilitate monitoring of health status.
(iv) Measurements of feed consumption should be determined weekly during the first 13 weeks of the study and at approximately monthly intervals thereafter unless health status or body weight changes dictate otherwise. Measurement of water consumption should be determined at the same intervals if the test substance is administered in the drinking water.
(v) Moribund animals shall be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. At the end of the study period, all survivors shall be sacrificed.
(8) Clinical pathology. At 12 months, 18 months, and at terminal sacrifice, a blood smear shall be obtained from all animals. A differential blood count should be performed on blood smears from those animals in the highest dosage group and the controls from the terminal sacrifice. If these data, or data from the pathological examination indicate a need, then the 12- and 18-month blood smears should also be examined. Differential blood counts should be performed for the next lower groups if there is a major discrepancy between the highest group and the controls. If clinical observations suggest a deterioration in health of the animals during the study, a differential blood count of the affected animals shall be performed.
(9) Gross necropsy. (i) A complete gross examination shall be performed on all animals, including those that died during the experiment or were killed in a moribund condition.
(ii) At least the liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, spleen, brain, and heart should be weighed wet as soon as possible after dissection to avoid drying. The lungs should be weighed if the test substance is administered by the inhalation route. The organs should be weighed from interim sacrifice animals as well as from at least 10 animals per sex per group at terminal sacrifice.
(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination.
(A) Digestive system.
( 1 ) Salivary glands.
( 2 ) Esophagus.
( 3 ) Stomach.
( 4 ) Duodenum.
( 5 ) Jejunum.
( 6 ) Ileum.
( 7 ) Cecum.
( 8 ) Colon.
( 9 ) Rectum.
( 10 ) Liver.
( 11 ) Pancreas.
( 12 ) Gallbladder (mice).
(B) Nervous system.
( 1 ) Brain (multiple sections).
( 2 ) Pituitary.
( 3 ) Peripheral nerves.
( 4 ) Spinal cord (three levels).
( 5 ) Eyes (retina, optic nerve).
(C) Glandular system.
( 1 ) Adrenals.
( 2 ) Parathyroids.
( 3 ) Thyroids.
(D) Respiratory system.
( 1 ) Trachea.
( 2 ) Lung.
( 3 ) Pharynx.
( 4 ) Larynx.
( 5 ) Nose.
(E) Cardiovascular/hematopoietic system.
( 1 ) Aorta (thoracic).
( 2 ) Heart.
( 3 ) Bone marrow.
( 4 ) Lymph nodes.
( 5 ) Spleen.
(F) Urogenital system.
( 1 ) Kidneys.
( 2 ) Urinary bladder.
( 3 ) Prostate.
( 4 ) Testes/epididymides.
( 5 ) Seminal vesicles.
( 6 ) Uterus.
( 7 ) Ovaries.
( 8 ) Female mammary gland.
(G) Other.
( 1 ) Skin.
( 2 ) All gross lesions and masses.
(iv) In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx, and paranasal sinuses should be examined and preserved. In dermal studies, skin from treated and adjacent control skin sites should be examined and preserved.
(v) Inflation of lungs and urinary bladder with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in inhalation studies is essential for appropriate and valid histopathological examination.
(vi) Information from clinical pathology, and other in-life data should be considered before microscopic examination, since they may provide significant guidance to the pathologist.
(10) Histopathology. (i) The following histopathology shall be performed:
(A) Full histopathology on the organs and tissues under paragraph (d)(9) (iii) of this section of all animals in the control and high dose groups and all animals that died or were killed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(ii) If the results show substantial alteration of the animal's normal life span, the induction of effects that might affect a neoplastic response, or other effects that might compromise the significance of the data, the next lower dose levels shall be examined as described in paragraph (d)(10)(i) of this section.
(iii) An attempt should be made to correlate gross observations with microscopic findings.
(iv) Tissues and organs designated for microscopic examination should be fixed in 10 percent buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming.
(e) Data and reporting —(1) Treatment of results. (i) Data shall be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions, and the percentage of animals displaying each type of lesion.
(ii) All observed results (quantitative and qualitative) shall be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance criteria shall be selected during the design of the study.
(2) Evaluation of study results. (i) The findings of a carcinogenicity study should be evaluated in conjunction with the findings of previous studies and considered in terms of the toxic effects, the necropsy and histopathological findings. The evaluation shall include the relationship between the dose of the test substance and the presence, incidence, and severity of abnormalities (including behavioral and clinical abnormalities), gross lesions, identified target organs, body weight changes, effects on mortality, and any other general or specific toxic effects.
(ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailablity of the test substance should be considered.
(iii) In order for a negative test to be acceptable, it must meet the following criteria: No more than 10% of any group is lost due to autolysis, cannibalism, or management problems; and survival in each group is no less than 50% at 15 months for mice and 18 months for rats. Survival should not fall below 25% at 18 months for mice and 24 months for rats.
(iv) The use of historical control data from an appropriate time period from the same testing laboratory (i.e., the incidence of tumors and other suspect lesions normally occurring under the same laboratory conditions and in the same strain of animals employed in the test) is helpful for assessing the significance of changes observed in the current study.
(3) Test report. (i) In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, the following specific information shall be reported. Both individual and summary data should be presented.
(A) Test substance characterization should include:
( 1 ) Chemical identification.
( 2 ) Lot or batch number.
( 3 ) Physical properties.
( 4 ) Purity/impurities.
( 5 ) Identification and composition of any vehicle used.
(B) Test system should contain data on:
( 1 ) Species and strain of animals used and rationale for selection if other than that recommended.
( 2 ) Age including body weight data and sex.
( 3 ) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods.
( 4 ) Identification of animal diet.
( 5 ) Acclimation period.
(C) Test procedure should include the following data:
( 1 ) Method of randomization used.
( 2 ) Full description of experimental design and procedure.
( 3 ) Dose regimen including levels, methods, and volume.
(4) Test results —(i) Group animal data. Tabulation of toxic response data by species, strain, sex, and exposure level for:
(A) Number of animals exposed.
(B) Number of animals showing signs of toxicity.
(C) Number of animals dying.
(ii) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals.
(A) Time of death during the study or whether animals survived to termination.
(B) Time of observation of each abnormal sign and its subsequent course.
(C) Body weight data.
(D) Feed and water consumption data, when collected.
(E) Results of clinical pathology and immunotoxicity screen when performed.
(F) Necropsy findings including absolute/relative organ weight data.
(G) Detailed description of all histopathological findings.
(H) Statistical treatment of results where appropriate.
(I) Historical control data.
(J) Achieved dose (mg/kg/day) as a time-weighted average if the test substance is administered in the diet or drinking water.
(iii) Inhalation studies. In addition, for inhalation studies the following shall be reported:
(A) Test conditions. The following exposure conditions shall be reported.
( 1 ) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulate and aerosols, method of conditioning air, treatment of exhaust air and the method of housing the animals in a test chamber.
( 2 ) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size should be described.
(B) Exposure data. These shall be tabulated and presented with mean values and a measure of variability (e.g. standard deviation) and should include:
( 1 ) Airflow rates through the inhalation equipment.
( 2 ) Temperature and humidity of air.
( 3 ) Actual (analytical or gravimetric) concentration in the breathing zone.
( 4 ) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
( 5 ) Particle size distribution, calculated MMAD and geometric standard deviation (GSD).
( 6 ) Explanation as to why the desired chamber concentration and/or particle size could not be achieved (if applicable) and the efforts taken to comply with this aspect of the sections.
(f) Quality assurance. A system shall be developed and maintained to assure and document adequate performance of laboratory staff and equipment. The study shall be conducted in compliance with 40 CFR part 792—Good Laboratory Practice Standards.
(g) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Benitz, K.F. Ed. Paget, G.E. Measurement of Chronic Toxicity. Methods of Toxicology (Blackwell, Oxford, 1970) pp. 82-131.
(2) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States. pp. 36-45 (1959, 3rd Printing 1975).
(3) Goldenthal, E.I. and D'Aguanno, W. Evaluation of Drugs, Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The Association of Food and Drug Officials of the United States. pp. 60-67 (1959, 3rd Printing 1975).
(4) Organisation for Economic Co-operation and Development. Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 451 Carcinogenicity Studies (Paris, 1981).
(5) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines. Journal of Environmental Pathology and Toxicology. 11:161-182 (1977).
(6) Page, N.P. Eds. Kraybill and Mehlman. Concepts of a Bioassay Program in Environmental Carcinogenesis. Vol.3. Advances in Modern Toxicology (Hemisphere, Washington, DC., 1977) pp. 87-171.
(7) Sontag, J.M. et al. Guidelines for Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1 United States Cancer Institute, Division of Cancer Control and Prevention, Carcinogenesis Bioassay Program (Bethesda, MD).
§ 799.9430   TSCA combined chronic toxicity/carcinogenicity.
(a) Scope. This section is intended to meet the testing requirements under section 4 of the Toxic Substances Control Act (TSCA). The objective of a combined chronic toxicity/carcinogenicity study is to determine the effects of a substance in a mammalian species following prolonged and repeated exposure. The application of this section should generate data which identify the majority of chronic and carcinogenicity effects and determine dose-response relationships. The design and conduct should allow for the detection of neoplastic effects and a determination of the carcinogenic potential as well as general toxicity, including neurological, physiological, biochemical, and hematological effects and exposure-related morphological (pathology) effects.
(b) Source. The source material used in developing this TSCA test guideline is the Office of Prevention, Pesticides, and Toxic Substances (OPPTS) harmonized test guideline 870.4300 (August 1998, final guideline). This source is available at the address in paragraph (h) of this section.
(c) Definitions. The following definitions apply to this section.
Carcinogenicity is the development of neoplastic lesions as a result of the repeated daily exposure of experimental animals to a chemical by the oral, dermal, or inhalation routes of exposure.
Chronic toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by the oral, dermal, or inhalation routes of exposure.
Cumulative toxicity is the adverse effects of repeated dose occurring as a result of prolonged action on, or increased concentration of, the administered test substance or its metabolites in susceptible tissues.
Dose in a combined chronic toxicity/carcinogenicity study is the amount of test substance administered via the oral, dermal, or inhalation routes for a period of up to 24 months. Dose is expressed as weight of the test substance per unit body weight of test animal (milligrams per kilogram), or as weight of the test substance in parts per million (ppm) in food or drinking water. When exposed via inhalation, dose is expressed as weight of the test substance per unit volume of air (milligrams per liter) or as parts per million per day. For dermal application, dose is expressed as weight of the test substance (grams, milligrams) per unit body weight of the test animal (milligrams per kilogram) or as weight of the substance per unit surface area (milligrams per square centimeter) per day.
No-observed-effects level (NOEL) is the maximum dose used in a study which produces no observed adverse effects. The NOEL is usually expressed in terms of the weight of a test substance given daily per unit weight of test animal (milligrams per kilogram per day).
Target organ is any organ of a test animal showing evidence of an effect induced by a test substance.
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg body weight (expected human exposure may indicate the need for a higher dose level), using the procedures described for this study, produces no observable toxic effects or if toxic effects would not be expected based upon data of structurally related compounds, then a full study using three dose levels might not be necessary.
(e) Test procedures —(1) Animal selection —(i) Species and strain. Preliminary studies providing data on acute, subchronic, and metabolic responses should have been carried out to permit an appropriate choice of animals (species and strain). As discussed in other guidelines, the mouse and rat have been most widely used for assessment of carcinogenic potential, while the rat and dog have been most often studied for chronic toxicity. For the combined chronic toxicity/carcinogenicity study via the oral and inhalation routes, the rat is the species of choice and for the dermal route, the mouse is species of choice. If other species are used, the tester must provide justification/reasoning for their selection. The strain selected should be susceptible to the carcinogenic or toxic effect of the class of substances being tested, if known, and provided it does not have a spontaneous background incidence too high for meaningful assessment. Commonly used laboratory strains must be employed.
(ii) Age/weight. (A) Testing must be started with young healthy animals as soon as possible after weaning and acclimatization.
(B) Dosing should generally begin no later than 8 weeks of age.
(C) At commencement of the study, the weight variation of animals used must be within 20% of the mean weight for each sex.
(D) Studies using prenatal or neonatal animals may be recommended under special conditions.
(iii) Sex. (A) Equal numbers of animals of each sex must be used at each dose level.
(B) Females must be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 100 rodents (50 males and 50 females) must be used at each dose level and concurrent control group. At least 20 additional rodents (10 males and 10 females) should be used for satellite dose groups and the satellite control group. The purpose of the satellite group is to allow for the evaluation of chronic toxicity after 12 months of exposure to the test substance.
(B) For a meaningful and valid statistical evaluation of long term exposure and for a valid interpretation of negative results, the number of animals in any group should not fall below 50% at 15 months in mice and 18 months in rats. Survival in any group should not fall below 25% at 18 months in mice and 24 months in rats.
(C) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.
(D) Each animal must be assigned a unique identification number. Dead animals (and their preserved organs) and tissues, and microscopic slides shall be identified by reference to the unique numbers assigned.
(v) Husbandry. (A) Animals may be group-caged by sex, but the number of animals per cage must not interfere with clear observation of each animal. The biological properties of the test substance or toxic effects (e.g., morbidity, excitability) may indicate a need for individual caging. Rodents should be housed individually in dermal studies and during exposure in inhalation studies.
(B) The temperature of the experimental animal rooms should be at 22 ±3 °C.
(C) The relative humidity of the experimental animal rooms should be 50 ±20%.
(D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark.
(E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure uniform distribution and adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Animals should be fed and watered ad libitum with food replaced at least weekly.
(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least five days is recommended.
(2) Control and test substances. (i) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed, it should not elicit toxic effects itself nor substantially alter the chemical or toxicological properties of the test substance. It is recommended that wherever possible the usage of an aqueous solution be considered first, followed by consideration of a solution in oil, and finally solution in other vehicles.
(ii) One lot of the test substance should be used throughout the duration of the study if possible, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound, and, if possible, the name and quantities of contaminants and impurities.
(iii) If the test or control substance is to be incorporated into feed or another vehicle, the period during which the test substance is stable in such a mixture should be determined prior to the initiation of the study. Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the appropriate concentration of the test or control substance is contained in the mixture.
(3) Control groups. A concurrent control group is required. This group should be an untreated or sham-treated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the toxic properties of the vehicle are not known or cannot be made available, both untreated and vehicle control groups are required.
(4) Dose levels and dose selection. (i) For risk assessment purposes, at least three dose levels must be used, in addition to the concurrent control group. Dose levels should be spaced to produce a gradation of effects. A rationale for the doses selected must be provided.
(ii) The highest dose level in rodents should elicit signs of toxicity without substantially altering the normal life span due to effects other than tumors. The highest dose should be determined based on the findings from a 90-day study to ensure that the dose used is adequate to assess the chronic toxicity and the carcinogenic potential of the test substance. Thus, the selection of the highest dose to be tested is dependent upon changes observed in several toxicological parameters in subchronic studies. The highest dose tested need not exceed 1,000 mg/kg/day.
(iii) The intermediate-dose levels should be spaced to produce a gradation of toxic effects.
(iv) The lowest-dose level should produce no evidence of toxicity.
(v) For skin carcinogenicity studies, when toxicity to the skin is a determining factor, the highest dose selected should not destroy the functional integrity of the skin, the intermediate doses should be a minimally irritating dose and the low dose should be the highest nonirritating dose.
(vi) The criteria for selecting the dose levels for skin carcinogenicity studies, based on gross and histopathologic dermal lesions, are as follows:
(A) Gross criteria for reaching the high dose:
( 1 ) Erythema (moderate).
( 2 ) Scaling.
( 3 ) Edema (mild).
( 4 ) Alopecia.
( 5 ) Thickening.
(B) Histologic criteria for reaching the high dose:
( 1 ) Epidermal hyperplasia.
( 2 ) Epidermal hyperkeratosis.
( 3 ) Epidermal parakeratosis.
( 4 ) Adnexal atrophy/hyperplasia.
( 5 ) Fibrosis.
( 6 ) Spongiosis (minimal-mild).
( 7 ) Epidermal edema (minimal-mild).
( 8 ) Dermal edema (minimal-moderate).
( 9 ) Inflammation (moderate).
(C) Gross criteria for exceeding the high dose:
( 1 ) Ulcers-fissures, exudate/crust (eschar), nonviable (dead) tissues.
( 2 ) Anything leading to destruction of the functional integrity of the epidermis (e.g., caking, fissuring, open sores, eschar).
(D) Histologic criteria for exceeding the high-dose:
( 1 ) Crust (interfollicular and follicular).
( 2 ) Microulcer.
( 3 ) Degeneration/necrosis (mild to moderate).
( 4 ) Epidermal edema (moderate to marked).
( 5 ) Dermal edema (marked).
( 6 ) Inflammation (marked).
(5) Administration of the test substance. The three main routes of administration are oral, dermal, and inhalation. The choice of the route of administration depends upon the physical and chemical characteristics of the test substance and the form typifying exposure in humans.
(i) Oral studies. If the test substance is administered by gavage, the animals are dosed with the test substance on a 7-day per week basis for a period of at least 18 months for mice and hamsters and 24 months for rats. However, based primarily on practical considerations, dosing by gavage on a 5-day per week basis is acceptable. If the test substance is administered in the drinking water or mixed in the diet, then exposure should be on a 7-day per week basis.
(ii) Dermal studies. (A) Preparation of animal skin. Shortly before testing, fur should be clipped from not less than 10% of the body surface area for application of the test substance. In order to dose approximately 10% of the body surface, the area starting at the scapulae (shoulders) to the wing of the ileum (hipbone) and half way down the flank on each side of the animal should be shaved. Shaving should be carried out approximately 24 hours before dosing. Repeated clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care should be taken to avoid abrading the skin which could alter its permeability.
(B) Preparation of test substance. Liquid test substances are generally used undiluted, except as indicated in paragraph (e)(4)(vi) of this section. Solids should be pulverized when possible. The substance should be moistened sufficiently with water or, when necessary, with a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on toxicity of, and penetration of the skin by, the test substance should be taken into account. The volume of application should be kept constant, e.g., less than 100 µL for the mouse and less than 300 µL for the rat. Different concentrations of test solution should be prepared for different dose levels.
(C) Administration of test substance. The duration of exposure should be at least 18 months for mice and hamsters and 24 months for rats. Ideally, the animals should be treated with test substance for at least 6 hours per day on a 7-day per week basis. However, based on practical considerations, application on a 5-day per week basis is acceptable. Dosing should be conducted at approximately the same time each day. The test substance must be applied uniformly over the treatment site. The surface area covered may be less for highly toxic substances. As much of the area should be covered with as thin and uniform a film as possible. For rats, the test substance may be held in contact with the skin with a porous gauze dressing and nonirritating tape if necessary. The test site should be further covered in a suitable manner to retain the gauze dressing plus test substance and to ensure that the animals cannot ingest the test substance. The application site should not be covered when the mouse is the species of choice. The test substance may be wiped from the skin after the 6-hour exposure period to prevent ingestion.
(iii) Inhalation studies. (A) The animals should be exposed to the test substance, for 6 hours per day on a 7-day per week basis, for a period of at least 18 months in mice and 24 months in rats. However, based primarily on practical considerations, exposure for 6 hours per day on a 5-day per week basis is acceptable.
(B) The animals must be tested in dynamic inhalation equipment designed to sustain a minimum air flow of 10 air changes per hour, an adequate oxygen content of at least 19%, and uniform conditions throughout the exposure chamber. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into surrounding areas. It is not normally necessary to measure chamber oxygen concentration if airflow is adequate.
(C) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. Where a whole body chamber is used, individual housing must be used to minimize crowding of the test animals and maximize their exposure to the test substance. To ensure stability of a chamber atmosphere, the total volume occupied by the test animals shall not exceed 5% of the volume of the test chamber. It is recommended, but not required, that nose-only or head-only exposure be used for aerosol studies in order to minimize oral exposures due to animals licking compound off their fur. The animals should be acclimated and heat stress minimized.
(D) The temperature at which the test is performed should be maintained at 22 ±2 °C. The relative humidity should be maintained between 40 to 60%, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable.
(E) The rate of air flow must be monitored continuously but recorded at least three times during the exposure.
(F) Temperature and humidity must be monitored continuously but should be recorded at least every 30 minutes.
(G) The actual concentrations of the test substance must be measured in the animal's breathing zone. During the exposure period, the actual concentrations of the test substance must be held as constant as practicable and monitored continuously or intermittently depending on the method of analysis. Chamber concentration may be measured using gravimetric or analytical methods as appropriate. If trial run measurements are reasonably consistent (±10% for liquid aerosol, gas, or vapor; ±20% for dry aerosol), then two measurements should be sufficient. If measurements are not consistent, three to four measurements should be taken. If there is some difficulty in measuring chamber analytical concentration due to precipitation, nonhomogeneous mixtures, volatile components, or other factors, additional analyses of inert components may be necessary.
(H) During the development of the generating system, particle size analysis must be performed to establish the stability of aerosol concentrations with respect to particle size. The mass median aerodynamic diameter (MMAD) particle size range should be between 1-3 µm. The particle size of hygroscopic materials should be small enough when dry to assure that the size of the swollen particle will still be within the 1-3 µm range. Measurements of aerodynamic particle size in the animal's breathing zone should be measured during a trial run. If MMAD values for each exposure level are within 10% of each other, then two measurements during the exposures should be sufficient. If pretest measurements are not within 10% of each other, three to four measurements should be taken.
(I) Feed must be withheld during exposure. Water may also be withheld during exposure.
(J) When the physical and chemical properties of the test substance show a low flash point or the test substance is otherwise known or thought to be explosive, care must be taken to avoid exposure level concentrations that could result in an exposure chamber explosion during the test.
(6) Observation period. (i) This time period must not be less than 24 months for rats and 18 months for mice, and ordinarily not longer than 30 months for rats and 24 months for mice. For longer time periods, and where any other species are used, consultation with the Agency in regard to the duration of the study is advised.
(ii) Animals in a satellite group to assess chronic toxicity should be observed for 12 months.
(7) Observation of animals. (i) Observations must be made at least twice each day for morbidity and mortality. Appropriate actions should be taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals). General clinical observations shall be made at least once a day, preferably at the same time each day, taking into consideration the peak period of anticipated effects after dosing. The clinical condition of the animal should be recorded.
(ii) A careful clinical examination must be made at least once weekly. Observations should be detailed and carefully recorded, preferably using explicity defined scales. Observations should include, but not be limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, including tremors and convulsions, changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength, and stereotypes or bizarre behavior (e.g., self-mutilation, walking backwards).
(iii) Signs of toxicity should be recorded as they are observed including the time of onset, degree and duration.
(iv) Body weights must be recorded individually for all animals once prior to administration of the test substance, once a week during the first 13 weeks of the study and at least once every 4 weeks thereafter unless signs of clinical toxicity suggest more frequent weighing to facilitate monitoring of health status.
(v) Measurements of feed consumption should be determined weekly during the first 13 weeks of the study and then at approximately monthly intervals unless health status or body weight changes dictate otherwise. Measurements of water consumption should be determined at the same intervals if the test material is administered in drinking water.
(vi) Moribund animals must be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. At the end of the study period, all survivors must be sacrificed. Animals in the satellite group must be sacrificed after 12 months of exposure to the test substance (interim sacrifice).
(8) Clinical pathology. Hematology, clinical chemistry and urinalyses must be performed from 10 animals per sex per group. The parameters should be examined at approximately 6 month intervals during the first 12 months of the study. If possible, these collections should be from the same animals at each interval. If hematological and biochemical effects are seen in the subchronic study, testing shall also be performed at 3 months. Overnight fasting of animals prior to blood sampling is recommended.
(i) Hematology. The recommended parameters are red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, white blood cell count, differential leukocyte count, platelet count, and a measure of clotting potential, such as prothrombin time or activated partial thromboplastin time.
(ii) Clinical chemistry. (A) Parameters which are considered appropriate to all studies are electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance and signs of clinical toxicity.
(B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total protein, and albumin. More than two hepatic enzymes, (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be measured. Measurements of addtional enzymes (of hepatic or other origin) and bile acids, may also be useful.
(iii) If a test chemical has an effect on the hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated.
(iv) Other determinations that should be carried out if the test chemical is known or suspected of affecting related measures include calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and cholinesterases.
(v) Urinalyses. Urinalysis for rodents must be performed at the end of the first year of the study using timed urine collection. Urinalysis determinations include: appearance, volume, osmolality or specific gravity, pH, protein, glucose, and blood/blood cells.
(9) Ophthalmological examination. Examinations must be made on all animals using an ophthalmoscope or an equivalent device prior to the administration of the test substance and at termination of the study on 10 animals per sex in the high-dose and control groups. If changes in eyes are detected, all animals must be examined.
(10) Gross necropsy. (i) A complete gross examination must be performed on all animals, including those which died during the experiment or were sacrificed in a moribund condition.
(ii) At least, the liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, spleen, brain, and heart should be trimmed and weighed wet, as soon as possible after dissection to avoid drying. The lungs should be weighed if the test substance is administered by the inhalation route. The organs should be weighed from interim sacrifice animals as well as from at least 10 animals per sex per group at terminal sacrifice.
(iii) The following organs and tissues, or representative samples thereof, must be preserved in a suitable medium for possible future histopathological examination:
(A) Digestive system—salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when present) .
(B) Nervous system—brain (multiple sections, including cerebrum, cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibial, preferably in close proximity to the muscle), spinal cord (three levels, cervical, mid-thoracic, and lumbar), eyes (retina, optic nerve).
(C) Glandular system—adrenals, parathyroid, thyroid.
(D) Respiratory system—trachea, lungs, pharynx, larynx, nose.
(E) Cardiovascular/Hematopoietic system—aorta, heart, bone marrow (and/or fresh aspirate), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), spleen.
(F) Urogenital system—kidneys, urinary bladder, prostate, testes, epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.
(G) Other—all gross lesions and masses, skin.
(iv) In inhalation studies, the entire respiratory tract, including nose, pharynx, larynx, and paranasal sinuses should be examined and preserved. In dermal studies, skin from treated and adjacent control skin sites should be examined and preserved.
(v) Inflation of lungs and urinary bladder with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in inhalation studies is essential for appropriate and valid histopathological examination.
(vi) Information from clinical pathology and other in-life data should be considered before microscopic examination, since these data may provide significant guidance to the pathologist.
(11) [Reserved]
(12) Histopathology. (i) The following histopathology must be performed:
(A) Full histopathology on the organs and tissues, listed in paragraph (e)(10)(iii) of this section of all animals in the control and high dose groups and of all animals that died or were sacrificed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(ii) If the results show substantial alteration of the animal's normal life span, the induction of effects that might affect a neoplastic response, or other effects that might compromise the significance of the data, the next lower levels should be examined fully as described in paragraph (e)(12)(i) of this section.
(iii) An attempt should be made to correlate gross observations with microscopic findings.
(iv) Tissues and organs designated for microscopic examination should be fixed in 10% buffered formalin or a recognized suitable fixative as soon as necropsy is performed and no less than 48 hours prior to trimming.
(f) Data and reporting —(1) Treatment of results. (i) Data must be summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion.
(ii) When applicable, all observed results, quantitative and qualitative, must be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance criteria should be selected during the design of the study.
(2) Evaluation of study results. (i) The findings of a combined chronic toxicity/carcinogenicity study should be evaluated in conjunction with the findings of previous studies and considered in terms of the toxic effects, the necropsy and histopathological findings. The evaluation must include the relationship between the dose of the test substance and the presence, incidence and severity of abnormalities (including behavioral and clinical abnormalities), gross lesions, identified target organs, body weight changes, effects on mortality and any other general or specific toxic effects.
(ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailablity of the test substance should be considered.
(iii) In order for a negative test to be acceptable, it should meet the following criteria—no more than 10% of any group is lost due to autolysis, cannibalism, or management problems, and survival in each group is no less than 50% at 15 months for mice and 18 months for rats. Survival should not fall below 25% at 18 months for mice and 24 months for rats.
(iv) The use of historical control data from an appropriate time period from the same testing laboratory (i.e, the incidence of tumors and other suspect lesions normally occurring under the same laboratory conditions and in the same strain of animals employed in the test) is helpful for assessing the significance of changes observed in the current study.
(3) Test report. (i) In addition to the reporting requirements specified under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart J, the following specific information must be reported:
(A) Test substance characterization should include:
( 1 ) Chemical identification.
( 2 ) Lot or batch number.
( 3 ) Physical properties.
( 4 ) Purity/impurities.
( 5 ) Identification and composition of any vehicle used.
(B) Test system should contain data on:
( 1 ) Species and strain of animals used and rationale for selection if other than that recommended.
( 2 ) Age including body weight data and sex.
( 3 ) Test environment including cage conditions, ambient temperature, humidity, and light/dark periods.
( 4 ) Identification of animal diet.
( 5 ) Acclimation period.
(C) Test procedure should include the following data:
( 1 ) Method of randomization used.
( 2 ) Full description of experimental design and procedure.
( 3 ) Dose regimen including levels, methods, and volume.
(4) Test results. (i) Group animal data. Tabulation of toxic response data by species, strain, sex, and exposure level for:
(A) Number of animals exposed.
(B) Number of animals showing signs of toxicity.
(C) Number of animals dying.
(ii) Individual animal data. Data should be presented as summary (group mean) as well as for individual animals.
(A) Time of death during the study or whether animals survived to termination.
(B) Time of observation of each abnormal sign and its subsequent course.
(C) Body weight data.
(D) Feed and water consumption data, when collected.
(E) Achieved dose (milligrams/kilogram body weight) as a time-weighed average is the test substance is administered in the diet or drinking water.
(F) Results of ophthalmological examination, when performed.
(G) Results of hematological tests performed.
(H) Results of clinical chemistry tests performed.
(I) Results of urinalysis tests performed.
(J) Results of observations made.
(K) Necropsy findings including absolute/relative organ weight data.
(L) Detailed description of all histopathological findings.
(M) Statistical treatment of results where appropriate.
(N) Historical control data.
(iii) In addition, for inhalation studies the following should be reported:
(A) Test conditions. The following exposure conditions must be reported.
( 1 ) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air and the method of housing the animals in a test chamber.
( 2 ) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size should be described.
(B) Exposure data. These must be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and should include:
( 1 ) Airflow rates through the inhalation equipment.
( 2 ) Temperature and humidity of air.
( 3 ) Actual (analytical or gravimetric) concentration in the breathing zone.
( 4 ) Nominal concentration (total amount of test substance fed into the inhalation equipment divided by volume of air).
( 5 ) Particle size distribution, and calculated MMAD and geometric standard deviation.
( 6 ) Explanation as to why the desired chamber concentration and/or particle size could not be achieved (if applicable) and the efforts taken to comply with this aspect of the guidelines.
(g) Quality control. A system must be developed and maintained to assure and document adequate performance of laboratory equipment. The study must be conducted in compliance with 40 CFR Part 792—Good Laborary Practice Standards.
(h) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.
(1) Benitz, K.F. Measurement of Chronic Toxicity. Methods of Toxicology. Ed. G.E. Paget. Blackwell, Oxford. pp. 82-131 (1970).
(2) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Leiter L.W., Tilson H.A., MacPhail, R.C. Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicol. Teratol. 13, 599-609. (1991)
(3) D'Aguanno, W. Drug Safety Evaluation—Pre-Clinical Considerations. Industrial Pharmacology: Neuroleptic. Vol. I, Ed. S. Fielding and H. Lal. Futura, Mt. Kisco, NY. pp. 317-332 (1974).
(4) Fitzhugh, O.G. Chronic Oral Toxicity, Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. The Association of Food and Drug Officials of the United States. pp. 36-45 (1959, 3rd Printing 1975).
(5) Goldenthal, E.I. and D'Aguanno, W. Evaluation of Drugs, Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics. The Association of Food and Drug Officials of the United States. pp. 60-67 (1959, 3rd Printing 1975).
(6) Organization for Economic Cooperation and Development. Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 453 Combined Chronic Toxicity/Carcinogenicity Studies, Paris. (1981).
(7) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines. Journal of Environmental Pathology and Toxicology 11:161-182 (1977).
(8) Page, N.P. Concepts of a Bioassay Program in Environmental Carcinogenesis, Advances in Modern Toxicology. Vol.3, Ed. Kraybill and Mehlman. Hemisphere, Washington, DC pp. 87-171 (1977)
(9) Sontag, J.M. et al. Guidelines for Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1 (Bethesda: United States Cancer Institute, Division of Cancer Control and Prevention, Carcinogenesis Bioassay Program.
(10) Summary of the EPA Workshop on Carcinogenesis Bioassay via the Dermal Route. EPA Report 50/6-89-002; 50/6-89-003. Washington, DC.
(11) The Atlas Of Dermal Lesions, EPA Report 20T-004, U.S Environmental Protection Agency, Washington, DC.