Working Group 5: Reproductive Toxicity 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 DRAFT FOR CONSULTATION 14.07.10 Draft Report on Alternative (Non-Animal) Methods for Cosmetics Testing: current status and future prospects – 2010: Chapter Reproductive Toxicity Compiled by Workgroup 14 July 2010 Sarah Adler1, Thomas Broschard2, Susanne Bremer3, Mark Cronin4, George Daston5, Elise Grignard3, Aldert Piersma6, Guillermo Repetto7 and Michael Schwarz8 Centre for Documentation and Evaluation of Alternatives to Animal Experiments (ZEBET), Federal Institute for Risk Assessment (BfR), Berlin, Germany; Merck KGaA, Darmstadt, Germany; Institute for Health & Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy; School of Pharmacy and Chemistry Liverpool, John Moores University, Liverpool, England; Miami Valley Innovation Center, The Procter and Gamble Company, Cincinnati, USA; Laboratory for Health Protection Research, National Institute for Public Health and the Environment RIVM, Bilthoven, The Netherlands; National Institute of Toxicology and Forensic Sciences, University Pablo de Olavide, Sevilla, Spain; Institute of Pharmacology und Toxicology, University of Tuebingen, Germany Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 34 Executive Summary 35 In the last decades, significant efforts have been undertaken to develop alternative methods to 36 assess reproductive toxicity However, despite the impressive number of alternative tests that 37 have been published and are listed in this report, the majority of these tests have not yet 38 gained regulatory acceptance There are a number of reasons for the relatively slow progress 39 in the implementation of alternative methods for reproductive toxicity safety evaluations, 40 these include: the lengthy research and development phase; a lack of understanding of the 41 mode of actions of reproductive toxicants; and the huge number of physiological mechanisms 42 involved in mammalian reproduction which can be affected by xenobiotics Among the 43 various stages in the reproductive cycle, embryo-foetal development is considered as one of 44 the most critical steps Substantial effort has been spent in the development of promising in 45 vitro assays, such as the Zebrafish embryo test and pluripotent embryonic stem cell models, to 46 allow for the detection of the teratogenic potential of substances However, besides their 47 current role as mechanistic support and screening tools, the role of alternative methods as part 48 of integrated testing strategies for regulatory toxicity evaluations has to be defined further 49 The complexity of mammalian reproduction requires integrated testing strategies to fulfil all 50 needs for hazard identification and risk assessment A promising way forward is the use of 51 recently established comprehensive databases in which toxicological information derived 52 from standardised animal experimentations is collected These databases will allow for the 53 identification of the most sensitive targets of reproductive toxicants This priority setting of 54 sensitive endpoints is the first step to obtain a detailed understanding of the toxicological 55 relevance of the in vitro tests described in this report and how they can be used in integrated 56 testing strategies Furthermore, this mapping exercise will also support the identification of 57 information gaps where further efforts in test development are necessary to design specific 58 alternative methods covering identified sensitive endpoints 59 According to the Cosmetics Directive 76/768/EEC only alternatives leading to full 60 replacement of animal experiments are of relevance for safety evaluations of cosmetic 61 ingredients Regardless, the retrospective analysis of available in vivo data allowing the 62 detection of the most sensitive endpoints, the definition of a tool-box of alternative methods 63 as well as the eventual need to develop additional alternatives to cover the missing building 64 blocks in the testing strategy will need more than 10 years to complete 65 66 67 68 Working Group 5: Reproductive Toxicity 69 70 DRAFT FOR CONSULTATION 14.07.10 Introduction 2.1 Complexity of the Reproductive Cycle 71 Reproductive toxicity refers to a wide variety of toxicological effects that may occur in 72 different phases within the reproductive cycle (figure 1) This includes effects on fertility, 73 sexual behaviour, embryo implantation, embryonic/foetal development, parturition, postnatal 74 adaptation, and subsequent growth and development into sexual maturity An enormous 75 variety of mechanisms at the molecular, cellular and tissue levels cooperate in a concerted and 76 genetically programmed way to regulate these processes The sensitivity to chemical insults 77 may differ extensively between processes In addition, different temporal windows of 78 sensitivity have been observed for different processes As an example, neural tube closure 79 occurs early in pregnancy, and most effects on this process can only be determined after 80 exposure during this critical period of time 81 82 83 Sexual maturation Growth and development Gamete production Postnatal development Fertilisation Transport of the zygote Birth Fetogenesis Implantation Embryogenesis 84 85 86 87 88 Figure 1: The main stages in the mammalian reproductive cycle 89 Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 90 91 92 2.2 Alternatives for Reproductive Toxicity Testing 93 Over the last two decades, a wealth of ex vivo and in vitro assays have been proposed as 94 alternative test systems for testing toxic effects on the various processes in reproduction and 95 development Individual in vitro models are reductionistic in nature and are therefore unable to 96 cover all aspects of the reproductive cycle since reproduction requires a complex interplay of 97 integrated functions However, parts of the reproductive cycle can be mimicked by in vitro 98 systems and it is conceivable that a panel of well-designed and validated in vitro tests could 99 replace a substantial proportion of in vivo testing procedures This chapter gives an inventory 100 of the current state of development of alternative test systems for reproductive toxicity hazard 101 assessment 102 Although not applicable for cosmetic ingredients, reduction of animal studies is a more 103 feasible goal than replacement, one example being the current OECD activity towards an 104 extended-1-generation study protocol, which, if it would replace the current 2-generation 105 study, would reduce animal use by roughly 40% in each study [1] The addition of relevant 106 parameters to this novel study protocol represents a good example of refined testing 107 108 Information Requirements for the Safety Assessment of Cosmetic 109 Within the EU the safety of cosmetic products is regulated by the Cosmetics Product 110 Directive 76/768/EEC [2] which will be replaced stepwise by the new EU Cosmetics 111 Regulation 1223/2009 According to Article of Directive 76/768/EEC, a “cosmetic product 112 put on the market must not cause damage to human health when applied under normal or 113 reasonably foreseeable conditions of use” In addition, Article 7a of the same Directive states 114 that the safety evaluation of a finished product should be based on the general toxicological 115 profile, the chemical structure and the level of exposure of each ingredient This implies that 116 a quantitative risk assessment is required for each single ingredient of a cosmetic product 117 Being responsible for the safety of its cosmetic product, the producer performs a risk 118 assessment based on the data of all ingredients used Therefore, a pre-market approval is not 119 necessary for most ingredients used for cosmetics However, certain ingredients listed in 120 positive lists of the Cosmetics Directive such as colorants (Annex IV), preservatives (Annex 121 VI), UV filters (Annex VII) and, most recently, hair dyes require approval of their safety prior 122 to marketing by the EU commission which require the submission of a full dossier[3] 123 Specific requirements for the evaluation of the safety of a cosmetic ingredient are not further Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 124 specified in the Directive either with regard to reproductive toxicity or to any other 125 toxicological endpoint However, information on data requirements with regard to the safety 126 evaluation of cosmetic ingredients are provided in the Notes of Guidance of the former SCCP 127 (now SCCS) [4]: For substances which are submitted for inclusion in the positive lists of the 128 Cosmetics Directive, a comprehensive dossier must be provided for evaluation by the SCCS 129 The dossier includes data on acute toxicity (if available), dermal and mucous membrane 130 irritation, dermal penetration, skin sensitization, repeated dose toxicity, genotoxicity, and 131 phototoxicity (if the cosmetic product is intended to be used on sunlight-exposed skin) 132 Further, it is stated that when considerable oral intake is expected, or when dermal penetration 133 data suggest a significant systemic absorption, information on toxicokinetics, carcinogenicity 134 and reproductive toxicity “may become necessary” Additional recommendations on specific 135 in vivo or in vitro reproductive toxicity studies to be submitted with a dossier are not described 136 in the Notes on Guidance From the SCCS/SCCP opinions published within recent years 137 (2000 – 2009) (http://ec.europa.eu/health/ph_risk/committees/04_sccs/sccs_opinions_en.htm) it can 138 be concluded that in most cases an in vivo developmental toxicity study in the rat (OECD TG 139 414) - submitted by the manufacturer as the only study on reproductive toxicity - was 140 considered sufficient by the SCCS In only a few cases additional data from a 1- or 2- 141 generation study (OECD TG 415 and 416) were included in a dossier [5] 142 For substances, which are not listed in one of the Annexes of the Cosmetic Directive, data on 143 reproductive toxicity are not explicitly asked for in the Notes of Guidance However, some 144 indications of adverse effects on the fertility could be obtained e.g from repeated dose 145 toxicity studies, if available (e.g histopathologic effects on reproductive organs, effects on the 146 endocrine system) 147 148 149 150 Inventory of Animal Test Methods Currently Used for the Evaluation of 151 Developmental and Reproductive Toxicity 152 In the following, OECD test guidelines for the regulatory investigation of the developmental 153 and reproductive toxicity of chemicals are described The list comprises an inventory of the 154 main study protocols However, not all of them are used for testing cosmetic ingredients 155 156 157 4.1 OECD Test Guideline 414: Prenatal Development Toxicity Study for the Testing of Chemicals 158 This Guideline provides general information concerning the effects of prenatal exposure on 159 the pregnant test animal and on the developing organism; this may include assessment of Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 160 maternal effects as well as death, structural abnormalities, or altered growth in the foetus The 161 guideline is not intended to examine solely the period of organogenesis, (e.g days 5-15 in the 162 rodent, and days 6-18 in the rabbit) but also effects from preimplantation, when appropriate, 163 through the entire period of gestation to the day before caesarean section Functional deficits, 164 although an important part of development, are not a part of this Guideline They may be 165 tested for in a separate study or as an adjunct to this study using the Guideline for 166 developmental neurotoxicity 167 The test substance is normally administered to pregnant animals at least from implantation to 168 one day prior to the day of scheduled kill, which should be as close as possible to the normal 169 day of delivery The Guideline is intended for use with rodent (preferably rat) and non-rodent 170 (preferably rabbit) Each test and control group should contain a sufficient number of females 171 to result in approximately 20 female animals with implantation sites at necropsy Three 172 concentrations, at least, should be used The test substance or vehicle is usually administered 173 orally by intubation A limit test may be performed if no effects are expected at a dose of 174 1000 mg/kg bw/d The study includes measurements (weighing) and clinical daily 175 observations One day prior to the expected day of delivery the females are killed, the uterine 176 contents are examined, and the foetuses are evaluated for soft tissue and skeletal changes In 177 any study which demonstrates an absence of toxic effects, further investigation to establish 178 absorption and bioavailability of the test substance should be considered [6] 179 180 4.2 OECD Test Guideline 415: One-Generation Reproduction Toxicity Study 181 This Test Guideline provides general information concerning the effects of a test substance on 182 male and female reproductive performance, such as gonadal function, oestrous cycle, mating 183 behaviour, conception, parturition, lactation and weaning The study may also provide 184 preliminary information about developmental toxic effects of the test substance, such as 185 neonatal morbidity, mortality, behaviour and teratogenesis and to serve as a guide for 186 subsequent tests The test substance is administered orally in graduated doses to several 187 groups of males and females 188 Males should be dosed during growth and for at least one complete spermatogenic cycle; 189 females of the Parent generation should be dosed for at least two complete oestrous cycles 190 The animals are then mated The test substance is administered to both sexes during the 191 mating period and thereafter only to females during pregnancy and for the duration of the 192 nursing period This Test Guideline is intended primarily for use with the rat or mouse Each 193 test and control group should contain a sufficient number of animals to yield about 20 Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 194 pregnant females at, or near, term Three test groups, at least, should be used It is 195 recommended that the test substance be administered in the diet or drinking water A limit test 196 may be performed if no effects would be expected at a dose of 1000 mg/kg bw/d The results 197 of this study include measurements (weighing, food consumption) and daily and detailed 198 observations, each day preferably at the same time, as well as gross necropsy and 199 histopathology The findings of a reproduction toxicity study should be evaluated in terms of 200 the observed effects, necropsy and microscopic findings [7] 201 202 4.3 OECD Test Guideline 416: Two-Generation Reproduction Toxicity 203 This Guideline provides general information concerning the effects of a substance on the 204 integrity and performance of the male and female reproductive systems, and on the growth 205 and development of the offspring, including gonadal function, the oestrus cycle, mating 206 behaviour, conception, gestation, parturition, lactation, and weaning, and the growth and 207 development of the offspring The study may also provide information about the effects on 208 neonatal morbidity, mortality, and preliminary data on prenatal and postnatal developmental 209 toxicity as well as serving as a guide for subsequent tests In addition to studying growth and 210 development of the F1 generation, this Guideline is also intended to assess the integrity and 211 performance of the male and female reproductive systems as well as growth and development 212 of the F2 generation For further information on developmental toxicity and functional 213 deficiencies, either additional study segments can be incorporated into this protocol, utilising 214 the Guidelines for developmental toxicity and/or developmental neurotoxicity, or these 215 endpoints could be studied in separate studies 216 The test substance is administered daily in graduated doses to several groups of males and 217 females Males and females of the Parent generation (5-9 weeks old) should be dosed during 218 growth, their mating, the resulting pregnancies and through the weaning of their first 219 generation offspring The administration of the substance is continued to first generation 220 offspring during their growth into adulthood, mating and production of a second generation 221 (until the weaning) The rat is the preferred species for testing Each test and control group 222 should contain a sufficient number of animals to yield preferably not less than 20 pregnant 223 females at or near parturition At least three dose levels and a concurrent control shall be used 224 A limit test may be performed if no effects would be expected at a dose of 1000 mg/kg bw/d 225 The results of this study include: measurements (weighing, sperm parameters, oestrus cycle 226 parameters and offspring parameters), clinical daily observations, as well as gross necropsy 227 and histopathology The findings of this two-generation reproduction toxicity study should be Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 228 evaluated in terms of the observed effects including necropsy and microscopic findings A 229 properly conducted reproductive toxicity test should provide a satisfactory estimation of a no- 230 effect level and an understanding of adverse effects on reproduction, parturition, lactation, 231 postnatal development including growth and sexual development [8] 232 233 4.4 OECD Test Guideline 421: Reproduction/Developmental Toxicity Screening Test 234 This Guideline generates limited information concerning the effects of a substance on male 235 and female reproductive performance such as gonadal function, mating behaviour, 236 conception, development of the conceptus and parturition It is not an alternative to, nor does 237 it replace the existing Test Guidelines 414, 415 and 416 This Screening Test Guideline can 238 be used to provide initial information on possible effects on reproduction and/or development 239 This test does not provide complete information on all aspects of reproduction and 240 development In particular, it offers only limited means of detecting post-natal manifestations 241 of prenatal exposure, or effects that may be induced during post-natal exposure Due (amongst 242 other reasons) to the relatively small numbers of animals in the dose groups, the selectivity of 243 the end points, and the short duration of the study, this method will not provide evidence for 244 definite claims of no effects However, positive results are useful for initial hazard assessment 245 and contribute to decisions with respect to the necessity and timing of additional testing 246 247 The test substance is administered in graduated doses to several groups of male and female 248 rats Males should be dosed for a minimum of four weeks Females should be dosed 249 throughout the study, so approximately 54 days It is recommended that each group be started 250 with at least 10 animals of each sex Generally, at least three test groups and a control group 251 should be used Dose levels may be based on information from acute toxicity tests or on 252 results from repeated dose studies The test substance is administered orally and daily The 253 limit test corresponds to one dose level of at least 1000 mg/kg body weight The results of this 254 study include measurements (weighing, food/water consumption) and daily and detailed 255 observations, preferably each day at the same time, as well as gross necropsy and 256 histopathology The findings of this toxicity study should be evaluated in terms of the 257 observed effects, necropsy and microscopic findings Because of the short period of treatment 258 of the male, the histopathology of the testis and epididymus must be considered, along with 259 the fertility data, when assessing male reproductive effects [9] 260 261 262 4.5 OECD Test Guideline 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 263 The test may be particularly useful as part of the initial screening for the assessment of 264 chemicals for which little or no toxicological information is available and can serve as an 265 alternative to conducting two separate tests for repeated dose toxicity (Guideline 407) and 266 reproduction/developmental toxicity (Guideline 421), respectively It can also be used as a 267 dose range finding study for more extensive reproduction/developmental studies, or when 268 otherwise considered relevant 269 The method comprises the basic repeated dose toxicity study that may be used for chemicals 270 on which a 90-day study is not warranted (e.g when the production volume does not exceed 271 certain limits) or as a preliminary study to a long-term study It further comprises a 272 reproduction/developmental toxicity screening test and, therefore, can also be used to provide 273 initial information on possible effects on male and female reproductive performance such as 274 gonadal function, mating behaviour, conception, development of the conceptus and 275 parturition, either at an early stage of assessing the toxicological properties of chemicals This 276 test does not provide complete information on all aspects of reproduction and development In 277 particular, it offers only limited means of detecting postnatal manifestations of prenatal 278 exposure, or effects that may be induced during postnatal exposure Due (amongst other 279 reasons) to the selectivity of the endpoints and the short duration of the study, this method 280 will not provide evidence for definite claims of no reproduction/developmental effects 281 Although, as a consequence, negative data not indicate absolute safety with respect to 282 reproduction and development, this information may provide some reassurance if actual 283 exposures were clearly less than the dose related to the No Observed Adverse Effect Level 284 (NOAEL) The Guideline also places emphasis on neurological and immunological effects 285 The test substance is administered in graduated doses to several groups of male and female 286 rats Males should be dosed for a minimum of four weeks; females should be dosed 287 throughout the study (approximately 54 days) Normally, matings of "one male to one female" 288 should be used in this study It is recommended that the test substance be administered orally 289 by gavage Each group should be started with at least 10 animals of each sex Generally at 290 least three test groups and a control group should be used Dose levels should be selected 291 taking into account any existing toxicity and (toxico-) kinetic data available The limit test 292 corresponds to one dose level of at least 1000 mg/kg body weight The results of this study 293 include measurements (weighing, food/water consumption) and daily detailed observations 294 (including sensory reactivity to stimuli), preferably each day at the same time, as well as gross 295 necropsy and histopathology The findings of this toxicity study should be evaluated in terms 296 of the observed effects, necropsy and microscopic findings The evaluation will include the Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 297 relationship between the dose of the test substance and the presence or absence of 298 observations Because of the short period of treatment of the male, the histopathology of the 299 testis and epididymus must be considered along with the fertility data, when assessing male 300 reproduction effects [10] 301 302 4.6 OECD Test Guideline 426: Developmental Neurotoxicity Study 303 The developmental neurotoxicity study provides information on the potential functional and 304 morphological effects on the developing nervous system of the offspring of repeated exposure 305 to a substance during in utero and early postnatal development 306 A developmental neurotoxicity study can be conducted as a separate study, incorporated into 307 a reproductive toxicity and/or adult neurotoxicity study (e.g., Test Guidelines 415, 416, 424), 308 or added onto a prenatal developmental toxicity study (e.g., Test Guideline 414) When the 309 developmental neurotoxicity study is incorporated within or attached to another study, it is 310 imperative to preserve the integrity of both study types 311 The test substance is administered daily, generally orally, to mated females (rats are preferred) 312 from the time of implantation (GD 6) throughout lactation (PND 21) At least three dose 313 levels and a concurrent control should be used and a total of 20 litters are recommended at 314 each dose level Dams are tested to assess effects in pregnant and lactating females and may 315 also provide comparative information Offspring are randomly selected from within litters for 316 neurotoxicity evaluation All dams and all offspring should be carefully observed at least once 317 daily with respect to their health, including morbidity and mortality The evaluation consists 318 of observations to detect gross neurological and behavioural abnormalities, and the evaluation 319 of brain weights and neuropathology during postnatal development and adulthood The report 320 should include the body weight, the food/water consumption; the detailed clinical 321 observations, the necropsy findings, a detailed description of all behavioural, the number of 322 animals at the start and at the end of the study and the toxic response data by sex and dose 323 level [11] 324 325 326 4.7 OECD Test Guideline 440: Uterotrophic Bioassay in Rodents: A short-term screening test for oestrogenic properties 327 The Uterotrophic Bioassay is an in vivo short-term screening test It evaluates the ability of a 328 chemical to elicit biological endocrine disruption activities consistent with agonists or 329 antagonists of natural oestrogens (e.g 17ß-estradiol) It is based on the increase in uterine 330 weight or uterotrophic response The uterus responds to oestrogens in two ways An initial 331 response is an increase in weight due to water imbibition This response is followed by a 10 Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 Covering different aspects of the steroid production incl gonadotrophinreceptors mediated toxicity Hazard identification and mechanistic studies Inhibition of androgens aromatisation Mechanistic studies Binding assays and reporter gene assays Mechanistic studies Optimisation > years Induction/inhibition of reporter gene product Mechanistic studies Optimisation 3-5 years OECD TG 416, Interaction with OECD TG 415 estrogen OECD TG 414 receptors OECD TG 440 Estrogen receptor binding, Induction of reporter gene product Mechanistic studies Validation and regulatory acceptance ongoing OECD TG 416, Interaction with OECD TG 415 Progesterone OECD TG 414 receptor Receptor binding, reporter gene assays Mechanistic studies Optimization 3-5 years OECD TG 416, Progesterone OECD TG 415 production OECD TG 414 Cell viability, progesterone production, histology Mechanistic studies R&D > years OECD TG 416, OECD TG 415 Other Steroidogenesis Assays OECD TG 416, OECD TG 415 Placental aromatase assay OECD TG 416, Thyroid OECD TG 415 receptor interactions OECD TG 416, Interaction with OECD TG 415 the aryl OECD TG 414 hydrocarbon receptor R&D > years Validation ongoing Different tests are based on various cell lines driving from rat, mouse and human tissues, as well as genetically engineered cells and primary leydig cells Different cellular assays as well as microsomal tests are in development 31 Working Group 5: Reproductive Toxicity OECD TG 416, Interaction with OECD TG 415 androgen OECD TG 414 receptor OECD TG 441 DRAFT FOR CONSULTATION 14.07.10 Binding and induction/inhibition of reporter gene product Mechanistic studies Optimization 3-5 years OECD TG 416, OECD TG 415 Gonadotrophin mediated effects release of gonadotrophins or receptor binding Human ex vivo model to assess transplacental Placenta transfer indicting perfusion system foetal exposure and metabolism of test chemicals OECD TG 416, Toxicity assessment Trophoblast cell OECD TG 415 during embryo assay OECD TG 414 implantation OECD TG 416, OECD TG 415 OECD TG 414 OECD TG 416, OECD TG 415 OECD TG 416, OECD TG 415 Mechanistic studies R&D Hazard identification that Optimisation might lead to fertility impairments R&D > years Several celluar tests based on primary cells/explants or cell lines covering FSH, GnRH, LH, induced effects are described 3-5 years > years Fertilisation, first Mouse embryo cleavage embryo bioassay development Hazard identification that Optimisation might lead to fertility impairments 3-5 years completion of oocyte meiosis up to the Follicle bioassay metaphase II (FBA) Oogenesis: oocyte yield; diameter; nuclear maturatio Hazard identification that Optimisation might lead to fertility impairments 3-5 years 32 Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 Bovine maturation test completion of oocyte meiosis up to the metaphase II Hazard identification that Optimisation might lead to fertility impairments Bovine fertilisation test OECD TG 416, OECD TG 415 Formation of female and male pronuclei after penetration of capacitated bull spermatozoa into matured oocytes Hazard identification that Optimisation might lead to fertility impairments 3-5 years DNA strand breaks and alkali labile sites in bull sperm Hazard identification that Optimisation might lead to fertility impairments 3-5 years Hazard identification that Optimisation might lead to fertility impairments 3-5 years Hazard identification that R&D might lead to fertility impairments > years OECD TG 416, OECD TG 415 OECD TG 416, OECD TG 415 ReProComet assay OECD TG 416, OECD TG 415 CASA test OECD TG 416, OECD TG 415 Sertoli cell tests multiple leading to impairments in motility, and viability as well as morphology of spermatozoa Secretion of Inhibin B by (1) primary rat Sertoli cells and (2) cells of line SerW3; cytotoxicity 3-5 years 33 Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 OECD TG 414 Stem cell based tests for assessing embryotoxicity OECD TG 414 OECD TG 414 OECD TG 414 OECD TG 414 Interaction with cell differentiation into neural, cartilage, bone and cardiac cells, chemical effects on signalling pathways Whole rat embryo culture Chicken embryo culture The Zebrafish Embryo Teratogenicity Assay Frog Embryo Teratogenesis Assay Xenopus (FETAX) OECD TG 416, Hamster Egg OECD TG 415 Penetration Test/ Hypoosmotic Swelling Test Assessment of lethality, malformations and growth retardation Assessment of lethality, malformations and growth retardation Functional status of the sperm: -sperm quality -fertilizing capacity Hazard identification of embryotoxicants Hazard identification of embryotoxicants Hazard identification of embryotoxicants Screening test for the hazard identification of developmental toxicants Screening test for the hazard identification of developmental toxicants Hazard identification leading to female and/or male toxicity Optimisation At least 3-5 years Stem cell based systems are difficult to optimized due to the challenges in stem cell differentiation protocols Validated Optimisation At least 3-5 years Optimisation At least 3-5 years Optimisation At least 3-5 years Optimisation A common standard protocol is needed to start prevalidation 3-5 years 951 34 Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 952 953 Identified Areas with no Alternative Methods Available and Related Scientific/ 954 Technical Difficulties 955 6.1 Approaches for alternative Testing 956 A significant number of alternative assays have been developed as described in chapter 957 However, their implementation in regulatory toxicity testing has not yet been achieved As 958 stated in the introduction to this chapter, the reproductive cycle combines a highly diverse 959 multitude of biological processes and mechanisms, each of which has their own time-related 960 sensitivity to xenobiotic exposures It is therefore a significant challenge to mimic all aspects 961 of the reproductive cycle with in vitro and in silico assays, which may be considered necessary 962 in order that reproductive toxicity can be predicted reliably on the basis of alternative assays 963 alone The classical aim of “one to one” replacement of in vivo protocols by alternative tests is 964 clearly not feasible for the complex reproductive and developmental toxicity animal study 965 protocols Alternative approaches are required in which a limited array of most sensitive 966 endpoints are reproduced by a set of alternative assays which, in combination, could provide 967 sufficient background for hazard identification and risk assessment other endpoints might be 968 identified for which alternative methods not yet exist These endpoints may include 969 amongst others ADME, spermatogenesis, sperm maturation, HPG axis and maternally 970 mediated effects 971 972 6.2 General limitations of in vitro methods for reproductive toxicity testing 973 It is generally recognised that in vitro methods represent only a very simplified picture of 974 reality, i.e of living organisms In the case of reproductive toxicity each in vitro model 975 encompasses only a small part of the complex reproductive cycle and not all steps of 976 reproduction are covered by those assays so far Moreover, many in vitro assays, e.g the 977 receptor binding assays, investigate rather a cellular mechanism which finally might or might 978 not result in an adverse effect in vivo Most in vitro assays not consider the various aspects 979 of absorption, distribution, metabolism and excretion (ADME), which have a tremendous 980 impact on the toxicological profile of a substance In addition, the distinction between general 981 toxicity and specific developmental effects is difficult to evaluate even with more complex in 982 vitro studies such as the whole embryo assays In general, the influence of the maternal 983 organism including maternal toxicity is not covered by in vitro studies Finally, technical 984 difficulties may occur such as low water solubility of test materials, which have to be 985 considered in the design of each individual in vitro assay 986 35 Working Group 5: Reproductive Toxicity 987 6.3 DRAFT FOR CONSULTATION 14.07.10 The Testing Strategy as the Future Driving Force 988 Classically, many in vitro alternatives have been developed based on relatively simple 989 endpoints that were deemed representative of the wider context of e.g embryotoxicity 990 Intrinsically reductionistic assays such as rodent whole embryo culture (WEC), the embryonic 991 stem cell test (EST), and the limb bud micromass (MM) were validated for their prediction of 992 the entire embryotoxicity endpoint This approach presumes that the endpoints represented in 993 these tests are actually among the critical ones for embryotoxicity prediction in general 994 Recent experience with the EST has shown that this is an oversimplification, leading to false 995 positives and false negatives and thus limited predictability, showing that the applicability 996 domain of the assay was more limited than anticipated This has led to the insight that rather 997 than focussing exclusively on individual assays and their relevance, it might be more 998 productive, in view of the regulatory implementation of alternative assays, to start from the 999 different direction, which is their anticipated role in testing strategies for chemical risk 1000 assessment 1001 1002 6.4 Retrospective Analyses to Select Critical Endpoints 1003 A wealth of in vivo data has been collected over the past 30 years since the introduction of 1004 OECD test guidelines for reproductive and developmental toxicity testing Databases 1005 collecting past experience are being built and allow detailed analysis to assess the relative 1006 sensitivity of endpoints in existing animal protocols [103] The combination of the most 1007 sensitive endpoints should be able to detect nearly all reproductive and developmental 1008 toxicants Current thinkings evolving towards designing alternative assays limited to covering 1009 each of these most sensitive endpoints only In addition, these assays should be fitted 1010 appropriately within the entire testing strategy either tiered, or as a battery, or in some 1011 combination of the two, in a phase where the information can be used optimally for hazard 1012 and risk assessment in order to preclude as much as possible any ultimate animal 1013 experimentation [104] 1014 1015 6.5 Towards the Definition of Novel Testing Paradigms 1016 The OECD conceptual framework for reproductive toxicity testing provides an outline of such 1017 an approach Starting from in silico (non-testing) information such as physico-chemical 1018 characteristics, structure-activity relationships and read-across methods, and followed by in 1019 vitro assays, it should be possible to restrict in vivo testing to an essential minimum Crucial 1020 steps in the process of designing the testing strategy on the basis of alternative approaches are 1021 the identification of most sensitive end points, evaluation of existing alternative tests 36 Working Group 5: Reproductive Toxicity DRAFT FOR CONSULTATION 14.07.10 1022 containing these end points, design of novel assays for sensitive endpoints not already 1023 addressed by current assays and optimisation of the testing strategy through the 1024 combination of assays It should be realized that the entire reproductive cycle in a living 1025 animal is, by definition, more than the sum of any combination of alternative approaches 1026 Therefore, for reproductive toxicity testing, animal studies will remain the last resort for 1027 foreseeable time This is caused by e.g the often long lag time between exposure and 1028 observed adverse effect in the reproductive cycle and also by kinetic aspects, which hamper 1029 ready translation from in vitro effective concentrations to in vivo effective doses In addition, 1030 efforts to build risk assessment solely on human derived data [105] are currently limited by 1031 the scarcity of relevant human toxicological data 1032 1033 6.6 Time Schedule for Phasing out in vivo Reproductive Toxicity Testing 1034 Given current knowledge and a realistic outlook into the future, full replacement of animal 1035 studies for reproductive toxicity hazard assessment is not probable within foreseeable time 1036 (>10 years) However, alternative assays are already being used for priority setting and 1037 screening purposes In addition, alternative assays can make an important contribution to the 1038 mechanistic understanding of reproductive toxicity Such tests may actually be able to give 1039 more specific information on the interference of the test compound with the endpoint 1040 involved than the in vivo study is able to generate The challenge is to build on these 1041 advantages of alternative tests in generating a testing strategy in which the most sensitive end 1042 points are combined in a well-informed selection of alternative assays Applying such a 1043 strategy in a tiered screening situation (cf the OECD conceptual framework) could preclude 1044 the in vivo testing of many reproductive toxicants and thus would considerably refine and 1045 reduce testing for reproductive toxicity 1046 1047 1048 1049 1050 1051 1052 1053 References [1] Cooper RL, Lamb JC, Barlow SM, Bentley K, Brady AM, Doerrer NG, 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complete information on all aspects of reproduction and 240 development In... Interaction with OECD TG 415 androgen OECD TG 414 receptor OECD TG 441 DRAFT FOR CONSULTATION 14.07.10 Binding and induction/inhibition of reporter gene product Mechanistic studies Optimization 3-5