(a) Purpose. In the assessment and evaluation of the toxic characteristics of a substance, determination of its potential to provoke skin sensitization reactions is important. Information derived from tests for skin sensitization serves to identify the possible hazard to a population repeatedly exposed to a test substance. While the desirability of skin sensitization testing is recognized, there are some real differences of opinion about the best method to use. The test selected should be a reliable screening procedure which should not fail to identify substances with significant allergenic potential, while at the same time avoiding false negative results.

(b) Definitions. (1) Skin sensitization (allergic contact dermatitis) is an immunologically mediated cutaneous reaction to a substance. In the human, the responses may be characterized by pruritis, erythema, edema, papules, vesicles, bullae, or a combination of these. In other species the reactions may differ and only erythema and edema may be seen.

(2) Induction period is a period of at least 1 week following a sensitization exposure during which a hypersensitive state is developed.

(3) Induction exposure is an experimental exposure of a subject to a test substance with the intention of inducing a hypersensitive state.

(4) Challenge exposure is an experimental exposure of a previously treated subject to a test substance following an induction period, to determine whether the subject will react in a hypersensitive manner.

(c) Principle of the test method. Following initial exposure(s) to a test substance, the animals are subsequently subjected, after a period of not less than 1 week, to a challenge exposure with the test substance to establish whether a hypersensitive state has been induced. Sensitization is determined by examining the reaction to the challenge exposure and comparing this reaction to that of the initial induction exposure.

(d) Test procedures. (1) Any of the following seven test methods is considered to be acceptable. It is realized, however, that the methods differ in their probability and degree of reaction to sensitizing substances.

(i) Freund's complete adjuvant test.

(ii) Guinea-pig maximization test.

(iii) Split adjuvant technique.

(iv) Buehler test.

(v) Open epicutaneous test.

(vi) Mauer optimization test.

(vii) Footpad technique in guinea pig.

(2) Removal of hair is by clipping, shaving, or possibly by depilation, depending on the test method used.

(3) Animal selection—(i) Species and strain. The young adult guinea pig is the preferred species. Commonly used laboratory strains should be employed. If other species are used, the tester should provide justification/reasoning for their selection.

(ii) Number and sex. (A) The number and sex of animals used will depend on the method employed.

(B) The females should be nulliparous and nonpregnant.

(4) Control animals. (i) Periodic use of a positive control substance with an acceptable level of reliability for the test system selected is recommended;

(ii) Animals may act as their own controls or groups of induced animals can be compared to groups which have received only a challenge exposure.

(5) Dose levels. The dose level will depend upon the method selected.

(6) Observation of animals. (i) Skin reactions should be graded and recorded after the challenge exposures at the time specified by the methodology selected. This is usually at 24, 48, and 72, hours. Additional notations should be made as necessary to fully describe unusual responses;

(ii) Regardless of method selected, initial and terminal body weights should be recorded.

(7) Procedures. The procedures to be used are those described by the methodology chosen.

(e) Data and reporting. (1) Data should be summarized in tabular form, showing for each individual animal the skin reaction, results of the induction exposure(s) and the challenge exposure(s) at times indicated by the method chosen. As a minimum, the erythema and edema should be graded and any unusual finding should be recorded.

(2) Evaluation of the results. The evaluation of results will provide information on the proportion of each group that became sensitized and the extent (slight, moderate, severe) of the sensitization reaction in each individual animal.

(3) Test report. In addition to the reporting requirements as specified under 40 CFR part 792, subpart J, the following specific information should be reported:

(i) A description of the method used and the commonly accepted name.

(ii) Information on the positive control study, including positive control used, method used, and time conducted.

(iii) The number and sex of the test animals.

(iv) Species and strain.

(v) Individual weights of the animals at the start of the test and at the conclusion of the test.

(vi) A brief description of the grading system.

(vii) Each reading made on each individual animal.

(f) References. For additional background information on this test guideline the following references should be consulted:

(1) Buehler, E.V. “Delayed Contact Hypersensitivity in the Guinea Pig,” Archives Dermatology. 91:171 (1965).

(2) Draize, J.H. “Dermal Toxicity,” Food Drug Cosmetic Law Journal. 10:722-732 (1955).

(3) Klecak, G. “Identification of Contact Allergens: Predictive Tests in Animals,” Advances in Modern Toxicology: Dermatology and Pharmacology. Ed. F.N. Marzulli and H.I. Maibach. (Washington, D.C.: Hemisphere Publishing Corp., 1977) 4:305-339).

(4) Klecak, G., Geleick, H., Grey, J.R. “Screening of Fragrance Materials for Allergenicity in the Guinea Pig.-1. Comparison of Four Testing Methods,” Journal of the Society of Cosmetic Chemists. 28:53-64 (1977).

(5) Magnusson, B., Kligman, A.M. “The Identification of Contact Allergens by Animal Assay,” The Guinea Pig Maximization Test. The Journal of Investigative Dermatology. 52:268-276 (1973).

(6) Maguire, H.C. “The Bioassay of Contact Allergens in the Guinea Pig” Journal of the Society of Cosmetic Chemists. 24:151-162 (1973).

(7) Maurer, T., Thomann, P., Weirich, E.G., Hess, R. “The Optimization Test in the Guinea Pig. A Method for the Predictive Evaluation of the Contact Allergenicity of Chemicals,” Agents and Actions. (Basel: Birkhauser Verlag, 1975) Vol. 5/2.

(8) Maurer, T., Thomann, P., Weirich, E.G., Hess, R. “The Optimization Test in the Guinea Pig: A Method for the Predictive Evaluation of the Contact Allergenicity of Chemicals,” International Congress Series Excerpta Medica No. 376, (1975) Vol. 203.

(a) Purpose. In the assessment and evaluation of the toxic characteristics of an inhalable material such as a gas, volatile substance, or aerosol/particulate, determination of the potential developmental toxicity is important. The inhalation developmental toxicity study is designed to provide information on the potential hazard to the unborn which may arise from exposure of the mother during pregnancy.

(b) Definitions. (1) Developmental toxicity is the property of a chemical that causes in utero death, structural or functional abnormalities or growth retardation during the period of development.

(2) “Aerodynamic diameter” applies to the behavioral size of particles of aerosols. It is the diameter of a sphere of unit density which behaves aerodynamically like the particles of the test substance. It is used to compare particles of different sizes, shapes, and densities and to predict where in the respiratory tract such particles may be deposited. This term is used in contrast to “optical,” “measured” or “geometric” diameters which are representation of actual diameters which in themselves cannot be related to deposition within the respiratory tract.

(3) “Geometric mean diameter” or “median diameter” is the calculated aerodynamic diameter which divides the particles of an aerosol in half based on the weight of the particles. Fifty percent of the particles by weight will be larger than the median diameter and 50 percent of the particles will be smaller than the median diameter. The median diameter and its geometeric standard deviation are used to statistically describe the particle size distribution of any aerosol based on the weight and size of the particles.

(4) “Inhalable diameter” refers to that aerodynamic diameter of a particle which is considered to be inhalable for the organism. It is used to refer to particles which are capable of being inhaled and may be deposited anywhere within the respiratory tract from the trachea to the deep lung (the alveoli). For man, the inhalable diameter is considered here as 15 micrometers or less.

(5) “Concentration” refers to an exposure level. Exposure is expressed as weight or volume of test substance per volume of air (mg/1), or as parts per million (ppm).

(6) “No-observed-effect level” is the maximum concentration in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of weight or volume of test substance given daily per unit volume of air.

(c) Principle of the test method. The test substance is administered in graduated concentrations, for at least that part of the pregnancy covering the major period of organogenesis, to several groups of pregnant experimental animals, one exposure level being used per group. Shortly before the expected date of delivery, the pregnant females are sacrificed, the uteri removed, and the contents examined for embryonic or fetal deaths, and live fetuses.

(d) Limit test. If a test at an exposure of 5 mg/1 (actual concentration of respirable substances) or, where this is not possible due to physical or chemical properties of the test substance, the maximum attainable concentration, produces no observable developmental toxicity, then a full study using three exposure levels might not be necessary.

(e) Test procedures—(1) Animal selection—(i) Species and strain. Testing shall be performed in at least two mamalian species. Commonly used species include the rat, mouse, rabbit, and hamster. If other mamalian species are used, the tester shall provide justification/reasoning for their selection. Commonly used laboratory strains shall be employed. The strain shall not have low fecundity and shall preferably be characterized for its sensitivity to developmental toxins.

(ii) Age. Young adult animals (nulliparous females) shall be used.

(iii) Sex. Pregnant female animals shall be used at each exposure level.

(iv) Number of animals. At least 20 pregnant rats, mice, or hamsters or 12 pregnant rabbits are required at each exposure level. The objective is to ensure that sufficient pups are produced to permit meaningful evaluation of the potential developmental toxicity of the test substance.

(2) Control group. A concurrent control group shall be used. This group shall be exposed to clean, filtered air under conditions identical to those used for the group exposed to the substance of interest. In addition, a vehicle-exposed group may be necessary when the substance under study requires a vehicle for delivery. It is recommended that during preliminary range finding studies, air vs. vehicle exposure be compared. If there is no substantial difference, air exposure itself would be an appropriate control. If vehicle and air exposure yield different results, both vehicle and air exposed control groups are recommended.

(3) Concentration levels and concentration selection. (i) At least three concentration levels with a control and, where appropriate, a vehicle control, shall be used.

(ii) The vehicle shall neither be developmentally toxic nor have effects on reproduction.

(iii) To select the appropriate concentration levels, a pilot or trial study may be advisable. Since pregnant animals have an increased minute ventilation as compared to non-pregnant animals, it is recommended that the trial study be conducted in pregnant animals. Similarly, since presumably the minute ventilation will vary with progression of pregnancy, the animals should be exposed during the same period of gestation as in the main study. In the trial study, the concentration producing embryonic or fetal lethalities or maternal toxicity should be determined.

(iv) Unless limited by the physical/chemical nature or biological properties of the substance, the highest concentration level shall induce some overt maternal toxicity such as reduced body weight or body weight gain, but not more than 10 percent maternal deaths.

(v) The lowest concentration level should not produce any grossly observable evidence of either maternal or developmental toxicity.

(vi) Ideally, the intermediate concentration level(s) shall produce minimal observable toxic effects. If more than one intermediate concentration is used, the concentration levels shall be spaced to produce a gradation of toxic effects.

(4) Exposure duration. The duration of exposure shall be at least six hours daily allowing appropriate additional time for chamber equilibrium.

(5) Observation period. Day 0 in the test is the day on which a vaginal plug and/or sperm are observed. The exposure period shall cover the period of major organogenesis. This may be taken as days 6 to 15 for rat and mouse, 6 to 14 for hamster, or 6 to 18 for rabbit.

(6) Inhalation exposure. (i)(A) The animals shall be tested in inhalation equipment designed to sustain a minimum dynamic air flow of 12 to 15 air changes per hour and ensure an adequate oxygen content of 19 percent and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. This is best accomplished by individual caging. To ensure stability of a chamber atmosphere, the total “volume” of the test animals shall not exceed 5 percent of the volume of the test chamber.

(B) Pregnant animals shall not be subjected to beyond the minimum amount of stress. Since whole-body exposure appears to be the least stressful mode of exposure, it is the method preferred. In general oro-nasal or head-only exposure, which is sometimes used to avoid concurrent exposure by the dermal or oral routes, is not recommended because of the associated stress accompanying the restraining of the animals. However, there may be specific instances where it may be more appropriate than whole-body exposure. The tester shall provide justification/reasoning for its selection.

(ii) A dynamic inhalation system with a suitable flow control system shall be used. The rate of air flow shall be adjusted to ensure that conditions throughout the exposure chamber are essentially the same. Test material distribution should be established before animals are committed to dosing. Maintenance of slight negative pressure inside the chamber will prevent leakage of the test substance into the surrounding areas.

(iii) The temperature at which the test is performed should be maintained at 22 °C (±2°) for rodents or 20 °C (±3°) for rabbits. Ideally, the relative humidity should be maintained between 40 to 60 percent, but in certain instances (e.g., tests of aerosols, use of water vehicle) this may not be practicable.

(7) Physical measurements. Measurements or monitoring should be made of the following:

(i) The rate of airflow shall be monitored continuously but shall be recorded at least every 30 minutes.

(ii) 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 shall be held as constant as practicable, monitored continously or intermittently depending on the method of analysis and measured at least at the beginning, at an intermediate time and at the end of the exposure period.

(iii) 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. During exposure, analysis shall be conducted as often as necessary to determine the consistency of particle size distribution.

(iv) Temperature and humidity shall be monitored continuously and be recorded at least every 30 minutes.

(8) Food and water during exposure period. Food should be withheld during exposure. Water may or may not be withheld. If it is not withheld it should not come in direct contact with the test atmospheres.

(9) Observation of animals. (i) A gross examination shall be made at least once each day.

(ii) Additional observations should be made daily with appropriate actions taken to minimize loss of animals to the study (e.g., necropsy or refrigeration of animals found dead and isolation or sacrifice of weak or moribund animals).

(iii) Signs of toxicity shall be recorded as they are observed, including the time of onset, the degree and duration.

(iv) Cage-side observations shall include, but not be limited to: Changes in skin and fur, eye and mucous membranes, as well as respiratory, autonomic and central nervous systems, somatomotor activity and behavioral pattern. Particular attention should be directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma.

(v) Measurements should be made weekly of food consumption for all animals in the study.

(vi) Animals shall be weighed at least weekly.

(vii) Females showing signs of abortion or premature delivery shall be sacrificed and subjected to a thorough macroscopic examination.

(10) Gross necropsy. (i) At the time of sacrifice or death during the study, the dam shall be examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.

(ii) Immediately after sacrifice or death, the uterus shall be removed, weighed, and the contents examined for embryonic or fetal deaths and the number of viable fetuses. Gravid uterine weights should not be obtained from dead animals if autolysis or where decomposition has occurred. The degree of resorption shall be described in order to help estimate the relative time of death.

(iii) The number of corpora lutea shall be determined for all species except mice.

(iv) The sex of the fetuses shall be determined and they shall be weighed individually, the weights recorded, and the mean fetal weight derived.

(v) Following removal, each fetus shall be examined externally.

(vi) For rats, mice and hamsters, one-third to one-half of each litter shall be prepared and examined for skeletal anomalies, and the remaining part of each litter shall be prepared and examined for soft tissue anomalies using appropriate methods.

(vii) For rabbits, each fetus shall be examined by careful dissection for visceral anomalies and then examined for skeletal anomalies.

(f) Data and reporting—(1) Treatment of results. 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 pregnant animals, the number and percentages of live fetuses and the number of fetuses with any soft tissue or skeletal abnormalities.

(2) Evaluation of results. The findings of a developmental toxicity study shall be evaluated in terms of the observed effects and the exposure levels producing effects. It is necessary to consider the historical developmental toxicity data on the species/strain tested. A properly conducted developmental toxicity study should provide a satisfactory estimation of a no-effect level.

(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:

(i) Test conditions. (A) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulates and aerosols, methods of conditioning air, and the method of housing the animals in a test chamber when this apparatus is used.

(B) The equipment for measuring temperature, humidity, and particulate aerosol concentrations and size shall be described.

(ii) Exposure data. These shall be tabulated and presented with mean values and a measure of variability (e.g., standard deviation) and shall include:

(A) Airflow rates through the inhalation equipment.

(B) Temperature of air.

(C) Nominal concentration—total amount of test substance fed into the inhalation equipment divided by volume of air (no standard deviation).

(D) Measured total concentrations (particulate and/or gaseous phases) in test breathing zone.

(E) Particle size distribution (e.g., median aerodynamic diameter of particles with geometric standard deviation) including estimates of the percents of inhalable and non-inhalable portions for the test animals.

(iii) Animal data. (A) Toxic response data by concentration.

(B) Species and strain.

(C) Date of death during the study or whether animals survived to termination.

(D) Date of onset and duration of each abnormal sign and its subsequent course.

(E) Feed, body weight and uterine weight data.

(F) Pregnancy and litter data.

(G) Fetal data (live/dead, sex, soft tissue and sketetal defects, resorptions).

(g) References. For additional background information on this test guideline the following references should be consulted:

(1) Department of Health and Welfare. The Testing of Chemicals for Carcinogenicity, Mutagenicity and Teratogenicity. Minister of Health and Welfare (Canada: Department of Health and Welfare, 1975).

(2) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances.” A report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).

(3) World Health Organization. Principles for the Testing of Drugs for Teratogenicity. WHO Technical Report Series No. 364. (Geneva: World Health Organization, 1967).

(a) Purpose. This guideline for two-generation reproduction testing is designed to provide general information concerning the effects of a test substance on gonadal function, conception, parturition, and the growth and development of the offspring. The study may also provide information about the effects of the test substance on neonatal morbidity, mortality, and preliminary data on teratogenesis and serve as a guide for subsequent tests.

(b) Principle of the test method. The test substance is administered to parental (P) animals prior to 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, up until the F2 generation is weaned.

(c) Test procedures—(1) Animal selection—(i) Species and strain. The rat is the preferred species. If another mammalian species is used, the tester shall provide justification/reasoning for its selection. Strains with low fecundity shall not be used.

(ii) Age. Parental (P) animals shall be about 5 to 8 weeks old at the start of dosing.

(iii) Sex. (A) For an adequate assessment of fertility, both males and females shall be studied.

(B) The females shall be nulliparous and non-pregnant.

(iv) Number of animals. Each test and control group shall contain at least 20 males and a sufficient number of females to yield at least 20 pregnant females at or near term.

(2) Control groups. (i) A concurrent control group shall be used. This group shall be an untreated or sham treated control group or if a vehicle is used in administering the test substance, a vehicle control group.

(ii) If a vehicle is used in administering the test substance, the control group shall receive the vehicle in the highest volume used.

(iii) If a vehicle or other additive is used to facilitate dosing, it shall not interfere significantly with absorption of the test substance or produce toxic effects.

(3) Dose levels and dose selection. (i) At least three dose levels and a concurrent control shall be used.

(ii) The highest dose level should induce toxicity but not high levels of mortality in the parental (P) animals.

(iii) The lowest dose level should not produce any grossly observable evidence of toxicity.

(iv) Ideally the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate dose is used, dose levels should be spaced to produce a gradation of toxic effects.

(4) Exposure conditions. The animals should be dosed with the test substance, ideally, on a 7 days per week basis.

(i) Dosing, mating, delivery, and sacrifice schedule.

(A) Daily dosing of the parental (P) males and females shall begin when they are 5 to 8 weeks old. For both sexes, dosing shall be continued for at least 10 weeks before the mating period.

(B) Dosing of P males shall continue through the 3 week mating period. At the end of the mating period, P males may be sacrificed and examined, or may be retained for possible production of a second litter. If these animals are retained for a second litter, dosing shall be continued. Dosing of the F1 males saved for mating shall continue from the time they are weaned through the period they are mated with the F1 females (11 weeks). F1 males may be sacrificed after the F1 mating period.

(C) Daily dosing of the P females shall continue through the three week mating period, pregnancy, and to the weaning of the F1 offspring. Dosing of the F1 females saved for mating shall continue from the time they are weaned, through the period they are mated with the F1 males (11 weeks from the time of weaning) pregnancy, and to the weaning of the F2 offspring.

(ii) All animals are sacrificed as scheduled.

(A) All P males should be sacrificed at the end of the 3-week mating period, or may be retained for possible production of a second litter. If these animals are retained for a second litter, dosing shall be continued.

(B) F1 males selected for mating should be sacrificed at the end of the three week period of the F1 mating.

(C) F1 males and females not selected for mating should be sacrified when weaned.

(D) The P females should be sacrificed upon weaning of their F1 offspring.

(E) F1 dams and their F2 offspring are sacrificed when the offspring are weaned.

(5) Administration of the test substance—(i) Oral studies. (A) It is recommended that the test substance be administered in the diet or drinking water.

(B) If administered by gavage or capsule, the dosage administered to each animal prior to mating shall be based on the individual animal's body weight and adjusted weekly. During pregnancy the dosage shall be based on the body weight at day 0 and 6 of pregnancy.

(ii) If another route of administration is used, the tester should provide justification and reasoning for its selection.

(6) Mating procedure—(i) Parental. (A) For each mating, each female shall be placed with a single male from the same dose level until pregnancy occurs or 1 week has elapsed. If mating has not occurred after 1 week, the female shall be placed with a different male. Paired matings should be clearly identified.

(B) Those pairs that fail to mate should be evaluated to determine the cause of the apparent infertility. This may involve such procedures as additional opportunities to mate with proven fertile males or females, histological examination of the reproductive organs, and examination of the estrus or spermatogenic cycles.

(C) Each day, the females shall be examined for presence of sperm or vaginal plugs. Day 0 of pregnancy is defined as the day vaginal plugs or sperm are found.

(ii) F1 cross. (A) For mating the F1 offspring, one male and one female are randomly selected at weaning from each litter for cross mating with another pup of the same dose level but different litter, to produce the F2 generation.

(B) F1 males and females not selected for mating are sacrificed upon weaning.

(iii) Special housing. After evidence of copulation, pregnant animals shall be caged separately in delivery or maternity cages. Pregnant animals shall be provided with nesting materials when parturition is near.

(iv) Standardization of litter sizes. (A) On day 4 after birth, the size of each litter should be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter.

(B) Whenever the number of male or female pups prevents having 4 of each sex per litter, partial adjustment (for example, 5 males and 3 females) is permitted. Adjustments are not appropriate for litters of less than 8 pups.

(C) Elimination of runts only is not appropriate.

(D) Adjustments of the F2 litters is conducted in the same manner.

(7) Observation of animals. (i) A gross examination shall be made at least once each day. Pertinent behavioral changes, signs of difficult or prolonged parturition, and all signs of toxicity, including mortality, shall be recorded. These observations shall be reported for each individual animal. Food consumption for all animals shall be monitored weekly except during the mating period.

(ii) The duration of gestation shall be calculated from day 0 of pregnancy.

(iii) Each litter should be examined as soon as possible after delivery for the number of pups, stillbirths, live births, sex, and the presence of gross anomalies. Live pups should be counted and litters weighed at birth or soon thereafter, and on days 4, 7, 14, and 21 after parturition.

(iv) Physical or behavioral abnormalities observed in the dams of offspring shall be recorded.

(v) P males and females shall be weighed on the first day of dosing and weekly thereafter. F1 litters shall be weighed at birth, or soon thereafter, and on days 4, 7, 14, and 21. In all cases, litter weights shall be calculated from the weights of the individual pups.

(8) Gross necropsy. (i) A complete gross examination shall be performed on all adult animals, including those which died during the experiment or were killed in moribund conditions.

(ii) Special attention shall be directed to the organs of the reproductive system.

(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: Vagina; uterus; ovaries; testes; epididymides; seminal vesicles; prostate, pituitary gland; and, target organ(s) when previously identified of all P and F1 animals selected for mating.

(9) Histopathology. Except if carried out in other studies of comparable duration and dose levels the following histopathology shall be performed:

(i) Full histopathology on the organs listed above for all high dose, and control P1 and F1 animals selected for mating.

(ii) Organs demonstrating pathology in these animals shall then be examined in animals from the other dose groups.

(iii) Microscopic examination shall be made of all tissues showing gross pathological changes.

(d) Data and reporting—(1) Treatment of results. 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 pregnant, the types of change and the percentage of animals displaying each type of change.

(2) Evaluation of study results. (i) An evaluation of test results, including the statistical analysis, based on the clinical findings, the gross necropsy findings, and the microscopic results shall be made and supplied. This should include an evaluation of the relationship, or lack thereof, between the animals' exposure to the test substance and the incidence and severity of all abnormalities.

(ii) 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:

(i) Toxic response data by sex and dose, including fertility, gestation, viability and lactation indices, and length of gestation.

(ii) Species and strain.

(iii) Date of death during the study or whether animals survived to termination.

(iv) Toxic or other effects on reproduction, offspring, or postnatal growth.

(v) Date of observation of each abnormal sign and its subsequent course.

(vi) Body weight data for P, F1, and F2 animals.

(vii) Necropsy findings.

(viii) Detailed description of all histopathological findings.

(ix) Statistical treatment of results where appropriate.

(e) References. For additional background information on this test guideline the following references should be consulted:

(1) Clermont, Y., Perry, B. “Quantitative Study of the Cell Population of the Seminiferous Tubules in Immature Rats,” American Journal of Anatomy. 100:241-267 (1957).

(2) Goldenthal, E.I. Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use. Drug Review Branch, Division of Toxicological Evaluation, Bureau of Science, Food and Drug Administration, Washington, DC (1966).

(3) Hasegawa, T., Hayashi, M., Ebling, F.J.G., Henderson, I.W. Fertility and Sterility. (New York: American Elsevier Publishing Co., Inc., 1973).

(4) Oakberg, E.F. “Duration of Spermatogenesis in the Mouse and Timing of Stages of the Cycle of the Seminiferous Epithelium,” American Journal of Anatomy. 9:507-516 (1956).

(5) Roosen-Runge, E.C. “The Process of Spermatogenesis in Mammals,” Biological Review. 37:343-377 (1962).

(a) Purpose. In the assessment and evaluation of the toxic characteristics of a chemical, determination of the potential developmental toxicity is important. The developmental toxicity study is designed to provide information on the potential hazard to the unborn which may arise from exposure of the mother during pregnancy.

(b) Definitions. (1) Developmental toxicity is the property of a chemical that causes in utero death, structural or functional abnormalities or growth retardation during the period of development.

(2) Dose is the amount of test substance administered. Dose is expressed as weight of test substance (g, mg) per unit weight of a test animal (e.g., mg/kg).

(3) No-observed-effect level is the maximum concentration in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of weight of test substance given daily per unit weight of test animal (mg/kg)

(c) Principle of the test method. The test substance is administered in graduated doses for at least part of the pregnancy covering the major period of organogenesis, to several groups of pregnant experimental animals, one dose level being used per group. Shortly before the expected date of delivery, the pregnant females are sacrificed, the uteri removed, and the contents examined for embryonic or fetal deaths, and live fetuses.

(d) Limit test. If a test at an exposure of at least 1000 mg/kg body weight, using the procedures described for this study, produces no observable developmental toxicity, then a full study using three dose levels might not be necessary.

(e) Test procedures—(1) Animal selection—(i) Species and strain. Testing shall be performed in at least 2 mammalian species. Commonly used species include the rat, mouse, rabbit, and hamster. If other mammalian species are used, the tester shall provide justification/reasoning for their selection. Commonly used laboratory strains shall be employed. The strain shall not have low fecundity and shall preferably be characterized for its sensitivity to developmental toxins.

(ii) Age. Young adult animals (nulliparous females) shall be used.

(iii) Sex. Pregnant female animals shall be used at each dose level.

(iv) Number of animals. At least 20 pregnant rats, mice or hamsters or 12 pregnant rabbits are required at each dose level. The objective is to ensure that sufficient pups are produced to permit meaningful evaluation of the potential developmental toxicity of the test substance.

(2) Control group. A concurrent control group shall be used. This group shall be an untreated or sham treated control group, or, if a vehicle is used in administering the test substance, a vehicle control group. Except for treatment with the test substance, animals in the control group(s) shall be handled in an identical manner to test group animals.

(3) Dose levels and dose selection. (i) At least 3 dose levels with a control and, where appropriate, a vehicle control, shall be used.

(ii) The vehicle shall neither be developmentally toxic nor have effects on reproduction.

(iii) To select the appropriate dose levels, a pilot or trial study may be advisable. It is not always necessary to carry out a trial study in pregnant animals. Comparison of the results from a trial study in non-pregnant, and the main study in pregnant animals will demonstrate if the test substance is more toxic in pregnant animals. If a trial study is carried out in pregnant animals, the dose producing embryonic or fetal lethalities or maternal toxicity shall be determined.

(iv) Unless limited by the physical/chemical nature or biological properties of the substance, the highest dose level shall induce some overt maternal toxicity such as reduced body weight or body weight gain, but not more than 10 percent maternal deaths.

(v) The lowest dose level should not produce any grossly observable evidence of either maternal or developmental toxicity.

(vi) Ideally, the intermediate dose level(s) should produce minimal observable toxic effects. If more than one intermediate concentration is used, the concentration levels should be spaced to produce a gradation of toxic effects.

(4) Observation period. Day 0 in the test is the day on which a vaginal plug and/or sperm are observed. The dose period shall cover the period of major organogenesis. This may be taken as days 6 to 15 for rat and mouse, 6 to 14 for hamster, or 6 to 18 for rabbit.

(5) Administration of test substance. The test substance or vehicle is usually administered orally, by oral intubation unless the chemical or physical characteristics of the test substance or pattern of human exposure suggest a more appropriate route of administration. The test substance shall be administered approximately the same time each day.

(6) Exposure conditions. The female test animals are treated with the test substance daily throughout the appropriate treatment period. When given by gavage, the dose may be based on the weight of the females at the start of substance administration, or, alternatively, in view of the rapid weight gain which takes place during pregnancy, the animals may be weighed periodically and the dosage based on the most recent weight determination.

(7) Observation of animals. (i) A gross examination shall be made at least once each day.

(ii) Additional observations shall be made daily with appropriate actions 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).

(iii) Signs of toxicity shall be recorded as they are observed, including the time of onset, the degree and duration.

(iv) Cage-side observations shall include, but not be limited to: changes in skin and fur, eye and mucous membranes, as well as respiratory, autonomic and central nervous systems, somatomotor activity and behavioral pattern.

(v) Measurements should be made weekly of food consumption for all animals in the study.

(vi) Animals shall be weighed at least weekly.

(vii) Females showing signs of abortion or premature delivery shall be sacrificed and subjected to a thorough macroscopic examination.

(8) Gross necropsy. (i) At the time of sacrifice or death during the study, the dam shall be examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.

(ii) Immediately after sacrifice or as soon as possible after death, the uterus shall be removed and the contents 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. The weight of the gravid uterus should be recorded for dams that are sacrificed. Gravid uterine weights should not be obtained from dead animals if autolysis or decomposition has occurred.

(iii) The number of corpora lutea shall be determined for all species except mice.

(iv) The sex of the fetuses shall be determined and they shall be weighed individually, the weights recorded, and the mean fetal weight derived.

(v) Following removal, each fetus shall be examined externally.

(vi) For rats, mice and hamsters, one-third to one-half of each litter shall be prepared and examined for skeletal anomalies, and the remaining part of each litter shall be prepared and examined for soft tissue anomalies using appropriate methods.

(vii) For rabbits, each fetus shall be examined by careful dissection for visceral anomalies and then examined for skeletal anomalies.

(f) Data and reporting—(1) Treatment of results. Data shall be summarized in tablular form, showing for each test group: the number of animals at the start of the test, the number of pregnant animals, the number and percentages of live fetuses and the number of fetuses with any soft tissue or skeletal abnormalities.

(2) Evaluation of results. The findings of a developmental toxicity study shall be evaluated in terms of the observed effects and the exposure levels producing effects. It is necessary to consider the historical developmental toxicity data on the species/strain tested. A properly conducted developmental toxicity study should provide a satisfactory estimation of a no-effect level.

(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:

(i) Toxic response data by concentration.

(ii) Species and strain.

(iii) Date of death during the study or whether animals survived to termination.

(iv) Date of onset and duration of each abnormal sign and its subsequent course.

(v) Food, body weight and uterine weight data.

(vi) Pregnancy and litter data.

(vii) Fetal data (live/dead, sex, soft tissue and skeletal defects, resorptions).

(g) References. For additional background information on this test guideline the following references should be consulted:

(1) Department of Health and Welfare. The Testing of Chemicals for Carcinogenicity, mutagenicity and Teratogenicity. Minister of Health and Welfare (Canada: Department of Health and Welfare, 1975).

(2) National Academy of Sciences. “Principles and Procedures for Evaluating the Toxicity of Household Substances.” A report prepared by the Committee for the Revision of NAS Publication 1138, under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC (1977).

(3) World Health Organization. Principles for the Testing of Drugs for Teratogenicity. WHO Technical Report Series No. 364. (Geneva: World Health Organization, (1967).