Toxicology Letters 226 (2014) 245–255

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Sensitivity of different generations and developmental stages in studies on reproductive toxicity F. Schulz ∗ , M. Batke, I. Mangelsdorf, C. Pohlenz-Michel, N. Simetska, G. Lewin Fraunhofer ITEM, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany

h i g h l i g h t s • • • •

Introduction of the new FeDTex database for prenatal development and reproductive toxicity studies. Analysis of the most responsive generation and developmental stage. Determination of the most affected critical targets in reproduction studies. Identification of F1 or F2 exclusive effects.

a r t i c l e

i n f o

Article history: Received 19 July 2013 Received in revised form 27 January 2014 Accepted 29 January 2014 Available online 10 February 2014 Keywords: FeDTex database Reproductive toxicology LOEL NOEL Risk assessment Multi-generation reproductive toxicity study

a b s t r a c t Numerous studies on reproductive toxicity are expected to be necessary under the EU program on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Therefore, it is important to analyse existing testing strategies including also the recently implemented extended one-generation reproduction toxicity study (EOGRTS, OECD guideline 443). For this purpose the responsiveness of the different generations and developmental stages in studies on reproductive toxicity is analysed and critical targets of reproductive toxicity are identified by using the Fraunhofer FeDTex database. The F1 generation is identified as most responsive generation in more than 50% of one-generation and multi-generation reproduction studies. Within the F1 generation the adult stage is mostly affected compared to the prenatal or postnatal stage. The target analysis in F1 has revealed alterations in body weight as highly sensitive for all developmental stages. Other important targets are the liver, kidney, testes, prostate, sperm parameters as well as developmental landmarks. The findings in the F2 generation have shown a higher responsiveness than F1 only in 3% of the studies. Although in 29 studies new effects are observed in F2 offspring compared to F1 irrespective of dose levels, overall no severe new effects have emerged that would change classification and labelling and justify an F1 mating. The presented data support the importance of F1 for risk assessment and demonstrate that the study design of the EOGRTS is a suitable alternative to two-generation studies. However, compared to a conventional one-generation study the EOGRTS may identify additional effects but will change risk assessment with respect to NOELs only in rare cases. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Currently, the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) program claims for the (re)evaluation of the toxicity of up to 100,000 chemicals until 2018 (Rovida and Hartung, 2009), including developmental and reproductive toxicity for industrial chemicals imported or manufactured at ≥10 tons per year according to mandatory endpoints mentioned in annexes VIII–X of the European REACH Regulation (EC, 2006). The required offspring studies are estimated to be responsible for

∗ Corresponding author. Tel.: +49 511 5350 318; fax: +49 511 5350 335.. E-mail address: fl[email protected] (F. Schulz). 0378-4274/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2014.01.045

approximately 90% of animal use and 70% of toxicity testing costs under REACH (Rovida and Hartung, 2009). Given the short time frame, this ambitious goal seems only feasible if existing data are utilised at their best, current testing strategies are optimised and new alternative in vitro and in silico methods are developed. This also contributes to the 3R-principle (Reduction, Refinement and Replacement of animal testing) originally published more than 50 years ago (Russell and Burch, 1959), primarily for ethical reasons but also due to cost savings and to allow a more rapid toxicity evaluation. Toxicity databases are integrated as useful tools into this process. The main task consists hereby in organising study data in an analysable format without losing information. Afterwards, the data pool can be used to analyse compound related toxicological properties and to refine toxicity testing as follows:

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Fig. 1. Scheme of effect data entries. Effect data entries are defined by the affected generation, subdivided into different developmental stages and corresponding organs or targets. The LOEL is documented gender-specifically. Examinations without effect result in a target-specific NOEL.

(I) Identification of critical targets in studies on reproductive and developmental toxicity to identify most responsive generations and developmental stages for (a) improvement of current in vivo testing strategies, (b) development of alternative in vitro methods and (c) identification of cellular level based adverse outcome pathways (AOPs). (II) Improvement and extension of current in silico models to predict the hazard of untested chemicals as trigger for the need of further testing or waiving of dispensable evaluations. Based on our recently developed FeDTex Database (Fertility and Developmental Toxicity in experimental animals database) critical targets in reproductive and developmental toxicity studies are identified and the most responsive generation and developmental stage in multi-generation reproduction studies are determined. The database also provides an extensive data pool for subsequent enhancement of in vitro and in silico models. 2. Materials and methods 2.1. Database structure of FeDTex The FeDTex DB was developed using Microsoft Access® and was integrated into a MySQLTM -based online platform. The database design distinguishes between three major parts: reference data, study design and toxicological data. The reference covers author, journal, volume and pages. Study design comprises general study data and provides major information on test substance, study type, species used including strain, sex and number of animals per dose group, exposure including dosage, route of application and duration, scope of examination and sacrifice. The toxicological data contain the results of the studies. Effects are assigned to associated targets/tissues and are characterised by their corresponding LOELs, differentiated to the affected developmental stage. For studies with an effect-free dose level the study NOEL is documented in the database. Examinations with no apparent effect on the target are documented additionally with their corresponding NOELs. Entry of toxicological data is described in Section 2.4. 2.2. Selection criteria for chemicals and studies FeDTex DB focuses on studies of organic compounds like industrial chemicals, pesticides, food additives and pharmaceuticals conducted in rodents (i.e. rat or mouse) and rabbits. Inorganic chemicals are included only to a minor extent. Metal compounds and mixtures as well as studies in other species are excluded. Prenatal development toxicity studies, one- and multi-generation reproduction studies (i.e. two- or three-generation reproduction studies and studies following the continuous breeding protocol) are generally accepted as study types. Oral and inhalation studies are preferred and represent more than 90% of the database content. Injection and dermal studies are included to a minor extent. All FeDTex DB entries are based on peer-reviewed publications. Common search engines like PubMed, Web of Science and SciFinder are used for literature research, in particular to screen for studies overlapping with the in-house database on repeated dose toxicity RepDose (Bitsch et al., 2006). To assure a suitable test design, e.g. duration of exposure, endpoints examined, number of dose levels tested, studies following OECD, U.S. EPA, ICH and/or Japanese MAFF guidelines are selected. To increase the amount of studies, studies with a comparable scope to guideline studies are additionally included.

2.3. Data entry standardisation To ensure consistent database entries and to facilitate queries for a comparative analysis of chemicals, study data and toxic effect data have to be standardised. Therefore, uniform glossaries are implemented into the database. Pick lists are notably available for the type of study, application route, species, strain, and examined generations. The treatment of animals and the scope of examination are further specified by unique tick-sheets. Information on treatment covers the affected sex, exposure concerning different life stages, and necropsies performed according to the developmental stage. Examinations are selected by setting of check marks for the respective generation. Additional information can be provided using free text fields. The toxicological effects and their related targets are also selected from corresponding pick lists. Furthermore, specific effects are attributed to their respective targets, therefore assuring a consistent data entry (i.e. the effect “hormone status (changed)” is solely available for the target “endocrine system”). The data entry standardisation is permanently validated and new terms can be added to the pick lists when necessary. 2.4. Toxicological data Effects are entered into FeDTex DB when statistical significance was proven, when a dose-response relationship was observed or the incidence was beyond the historical control range. Adverse and non-adverse effects are not distinguished. Thus the database provides NOELs and LOELs. All entries are cross-checked by the four-eye principle. Debatable effects (e.g. effects lacking a clear dose-response relationship) are labelled with a specific flag. This provides the opportunity to exclude these effects from evaluation. The effects finally entered into FeDTex DB follow a specific organisation chart (Fig. 1) and are dependent of the corresponding generation (i.e. F0, F1, F2 or F3), developmental stage (i.e. prenatal, postnatal up to puberty or adult), and target/organ. The prenatal stage covers foetal assessment and birth weight as markers of prenatal development. The postnatal stage covers all following examinations after birth up to puberty. Every effect is finally characterised by a specific LOEL. To be able to assess different susceptibility of the sexes, LOELs are provided for both sexes. As different effects can occur at a distinct target/organ, the target/organ LOEL is defined by the lowest effect LOEL in this target/organ and is documented in the database. A LOEL for each developmental stage and generation as well as an overall study LOEL is analogically generated and documented. Examinations without detected effect lead to a corresponding NOEL. 2.5. Comparison of FedTex DB and ToxRefDB data To compare the content of FeDTexDB with the Toxicity Reference Database (ToxRefDB), the latest available ToxRefDB-version (i.e. toxrefdb 2010q1b) from the U.S. EPA homepage was used for analysis. 2.6. Analysing the chemical domain of FeDTex DB using the QSAR Toolbox The chemical domain of FeDTex DB was analysed by using the OECD QSAR Toolbox V2.3. The Toolbox is an open source software intended to be used for grouping approaches such as read across and category definition. Several grouping tools are provided. It is possible to group according to (1) predefined groups such as categories derived from the US EPA New chemical or the OECD HPV program; (2) mechanistic aspects e.g. DNA binding or biodegradation; (3) endpoint specific aspects e.g. based on a certain reactivity observed in in vitro/in vivo assays; and (4) empiric methods e.g. chemical elements or organic functional groups. The substances of the FeDTex DB were grouped by using the organic functional group (OFG) profile provided in the Toolbox. The profiling system allows a classification of the characteristic structural fragments and different functionalities of organic chemicals and can be used to identify structurally similar chemicals. As substances may contain several functional groups, one single substance may also be assigned to more than one OFG.

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Table 1 Overview I: number and percentage of chemicals and studies covered by FeDTex DB.

Total Study type

Species

Route

Prenatal development toxicity One-generation reproduction Two-generation reproduction Three-generation reproduction Continuous breeding protocol Other Rat Mouse Rabbit Gavage Diet Drinking water Inhalation Injection Dermal

No. of chemicals

Pct. of chemicals

No. of studies

Pct. of studies

269 147 87 107 13 21 7 250 44 71 126 64 35 77 17 5

100 55 32 40 5 8 3 93 16 26 47 24 13 29 6 2

535 259 116 113 15 24 8 382 73 80 242 75 42 141 28 7

100 48 22 21 3 4 2 71 14 15 45 14 8 26 5 1

2.7. Determination of responsive generations and developmental stages

3. Results

The responsiveness of the different generations and the different developmental stages was analysed either by quantitatively comparing the dose levels of the respective NOEL or by qualitatively comparing the observed effects. Within the quantitative comparison (i.e. F0/F1 and F1/F2 or prenatal/postnatal, prenatal/adult and postnatal/adult) it was further distinguished between equally responsive generations or stages with NOELs at the same dose level (i.e. resulting in a dose level ratio equal to 1) and cases with one or the other generation/stage being more responsive, resulting in a dose level ratio above or below 1. Prior to the analysis of generation dose level ratios studies were selected based on 4 criteria (the number of excluded studies per criteria is provided in Supplementary 2):

3.1. Current database status

(a) In few reports the effects for a particular generation might not be documented or a following generation might be skipped during the study course (e.g. due to excessive mortality). To exclude these studies from evaluation, only studies with at least one examination performed (i.e. at least one LOEL or one NOEL present) in each generation were analysed. (b) Studies with a one-dose treatment were excluded from the evaluation as these studies may result in a vague data evaluation. Depending on the observed effects it is unclear, if the parental or the offspring generation had responded first or if the dose setting was correct at all. (c) Studies with no observed effects at all were excluded from the evaluation. In these studies, the dose levels cannot be presumed to be set correctly and it is unclear if the parental or the offspring generation had responded first and at which dose. (d) Studies lacking a NOEL in both compared generations (i.e. F0 and F1 in prenatal development and one-generation studies as well as F0 and F1 or F1 and F2 in multi-generation studies) were further excluded from the evaluation. Here, the dose setting was too high and it is unclear which generation might have responded first at lower doses. In contrast, if only one generation did not reveal a NOEL, it was still possible to consider the other generation as less responsive.

In conclusion, 93 multi-generation studies were included in the F0/F1- and 101 studies in the F1/F2-comparison. Furthermore, the F0/F1-generation ratio of 69 one-generation studies and as many as 208 developmental toxicity studies were evaluated. For the comparison of the different developmental stages of the F1-generation, studies were selected by the criteria that at least two dose groups were present and at least one examination was performed in each developmental stage (i.e. at least one LOEL or NOEL was present). Based on these criteria 169 studies were identified including 103 two-generation reproduction studies, 35 one-generation studies, 15 three-generation reproduction studies and 16 studies following the continuous breeding protocol.

At the time of evaluation the FeDTex DB contained toxicological data derived from 535 animal studies on 269 chemicals (Table 1). The structurally diverse chemical domain of the FeDTex DB is represented by 80 different chemical structures identified by the organic functional groups profile of the QSAR Toolbox V2.3 (data not shown). Within these structures frequent and reactive groups are identified as arenes (38%), alcohols (29%), ethers (24%), heterocyclic fragments (15%), carboxylic acids (10%), esters (8%) and secondary aliphatic amines (7%) among others. It has to be kept in mind that a single molecule can exhibit different functional groups and is thus assigned to different groups. For instance, the ethers comprise 17 glycolethers and 12 phthalates, the latter are also included in the carboxylic acid esters. Most of the included studies were published in the past three decades with a comparable count of about 170 studies per decade. About half of the FeDTex DB studies are prenatal development toxicity studies following OECD guideline 414 or a comparable study protocol. The other half are reproduction toxicity studies, mainly one-generation reproduction studies including developmental neurotoxicity studies (OECD guidelines 415, 426 or similar) and two-generation reproduction studies (OECD guideline 416 or similar). Furthermore, prenatal development toxicity studies cover more than one half, two-generation reproduction studies nearly 40% and one-generation reproduction studies cover about one third of the 269 inserted chemicals. Most studies were conducted in rats, which is also the preferred species in reproductive toxicology studies following OECD guidelines (OECD, 2013b). Studies in rabbits and mice together cover nearly one third of the study content. Treatment of animals was mainly carried out via oral application routes, as preferred in the guidelines, or via inhalation. A single chemical may be represented by different studies, study types, species, and/or routes of exposure (Table 2). About one half of the chemicals are covered by more than one study. The maximum study count for a single chemical is 10.

3.2. Data overlap with ToxRefDB 2.8. Qualitative comparison of effects in F1 and F2 For this comparison, all documented effects for F1 (N = 1400) and F2 (N = 462) derived from multi-generation studies including an F2 generation (N = 138) were evaluated. In case an effect was found in only one generation, it was further specified, if the effect was covered by similar effects or in a different developmental stage in the other generation. Coverage by additional parameters was taken as existent if the same qualitative conclusion could be drawn by other effects (e.g. increased prenatal mortality could also be described with an increased post-implantation loss).

ToxRefDB developed within the U.S. EPA’s ToxCast program contains data from multi-generation reproduction studies in rats (Martin et al., 2009) and prenatal development toxicity studies in rats and rabbits (Knudsen et al., 2009). CAS numbers of chemicals covered by FeDTex DB and ToxRefDB were analysed for a potential data overlap. In total only 6% of the chemicals covered by FeDTex DB overlap with chemicals contained in ToxRefDB (Table 3). When

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Table 2 Overview II: number and percentage of chemicals referring to different study parameters evaluated per single chemical.

Study count

Study types

Species

Routes

No. of different types per single chemical

No. of chemicals

Pct. of chemicals

1 2 3 4 ≥5 1 2 3 4 5 1 2 3 1 2 3 4

136 71 30 16 16 186 56 25 1 1 187 68 14 223 38 7 1

51 26 11 6 6 69 21 9 prenatal based on the data provided. 3.5. Targets determining the LOEL of the F1 generation The targets affected at the lowest LOEL of the F1 generation were analysed based on the 169 studies described before, likewise reflecting the impact of the different developmental stages on the F1 LOEL. Changes in body weight as sign of general toxicity are most frequently affected at the F1 LOEL in each developmental stage (Table 7). The major body weight influence is observed at the postnatal stage with nearly one third of the studies affected, followed by body weight changes in adults and altered foetal or birth weight at the prenatal stage. Clinical symptoms observed at the adult stage also strongly influence the F1 LOEL. Among a total of 33 effects assigned to clinical symptoms at the F1 LOEL, 70% consist of an altered food (15 effects) or water consumption (8 effects, data not shown). All but two of these effects were seen in adult F1 animals. Besides these general parameters, organ toxicity (i.e. weight changes, necropsy and histopathological findings) seems to be most important in adults, as organ weight determination in the early postnatal time frame is difficult and necropsies are rarely performed before weaning. Liver and kidney are most frequently affected but also alterations of the reproductive organs determine the F1 LOEL to a remarkable extent. Overall, the male reproductive targets (mainly testes and prostate) as well as sperm parameters are more frequently affected than the female reproductive

Table 4 Comparison of NOEL ratios among parental/offspring generations. Study type

Generation ratio

Prenatal development toxicity One-generation reproduction Multi-generation reproduction

F0/F1 F0/F1 F0/F1 F1/F2

Pct. of studies with a NOEL ratio:

N

1

45 23 21 70

25 20 26 20

30 57 53 10

208 69 93 101

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Table 5 Analysis of effects at the LOEL in studies with a lower NOEL in F2 compared to F1. Study no.

Effect in F2

Evaluation

LOELs [mmol/kg bw/day]

1

Birth weight increased

2

Pup body weight increased

3

Decreased mean water consumption

-No dose dependency -Effect not seen PND 4 or later -Effect not adverse -Only in F2b at weaning (no effect in F2a) -No dose dependency -Effect not adverse Same effect and LOEL in F0

4

Mortality increased (PND1)

5

Female anogenital distance increased

6

Male retention of nipples/areolae

-Dose dependent -Same LOEL as F0

7

Pup body weight decreased

Higher sensitivity of F0

8

Increased pup postnatal mortality and decreased pup body weight

-F2 most sensitive generation -Decreased fertility of F0 as trigger for mating of F1

9

Decreased pup body weight

10

Structural abnormalities in bone and kidneys pointing to growth retardation

-F2 most sensitive generation -Effect not considered as adverse -Effect occurring at higher dose level in F1 -Skeletal variations pointing to growth retardation observed in F1 foetuses -Dose dependent -F2 most sensitive generation -No trigger for mating of F1

F0: 0.45 F1: 0.45 F2: 0.14 F0: 0.60 F1: 0.60 F2: 0.07 F0: 0.17 F1: 0.34 F2: 0.17 F0: 0.05 F1: 0.11 F2: 0.05 F0: 3.28 × 10−6 F1: 0.16 F2: 3.28 × 10−6 F0: 0.007 F1: 0.035 F2: 0.007 F0: 11.04 F1: no effects F2: 55.22 F0: 0.14 F1: 0.14 F2: 0.07 F0: no effects F1: 13.25 F2: 3.31

-No dose dependency, mid dose only -No effect on lactation index -Same LOEL as F0 -Dose dependent, but not statistically significant at all doses -Same LOEL as F0

organs or fertility (i.e. decreased fertility index). Compared to adults organ toxicity is less frequently observed at the postnatal stage, most likely based on the currently limited guideline requirements. However, highly affected targets at the postnatal stage are predominantly developmental landmarks (i.e. eye opening, pinna detachment) and hormone regulated parameters (i.e. anogenital distance, retention of nipples, time and body weight at vaginal opening and preputial separation, and testicular descent) as well as an altered reflex ontogenesis. The endocrine system (i.e. changed hormone status or (onset of) oestrus cyclicity) is a noteworthy target for both postnatal and adult stage. Interestingly, the percentages of LOEL determining targets at the prenatal stage are comparatively low. Even if the percentages for all targets would be summed up, the total value is 20%, reflecting that in many of the analysed studies no toxicological effect at all is observed at the prenatal stage. Only mortality, often presented as an altered litter size, and skeletal effects should be mentioned. Other targets appear negligible. 3.6. Targets determining the F1 LOEL- considering the scope of examination As described in Section 2, the studies entered into FeDTex DB come from publications in the open literature. Not all studies were performed according to guidelines. On these grounds relevant Table 6 Comparison of NOEL ratios for the different developmental stages in studies on reproductive toxicity. Stage ratio

Prenatal/postnatal Prenatal/adult Postnatal/adult

Pct. of studies with a NOEL ratio: 1

8 9 18

28 19 43

64 72 39

N

169 169 169

F0: 0.33 F1: 0.33 F2: 0.08

targets might not have been investigated in the respective studies and thus are underestimated in the results of Table 7. Furthermore, guideline requirements changed substantially over the last 20 years. Hence, the influence of the scope of examination on the frequency of the different targets at the F1 generation LOEL was analysed. Since this evaluation is complex, only targets allowing a clear-cut matching between the scope of examination and the toxicological effects were analysed. Concerning organ toxicity only weight changes were considered in Table 8, as only this parameter allows a clear-cut comparison between the scope of examination and the observed effects. Despite the importance of macroscopic alterations, necropsy findings and histopathological changes were excluded in this evaluation. For these parameters the scope of examination is frequently inadequately described in publications. For instance, it may be stated that histopathology was performed, but the organs were not provided. On the other hand, to distinguish between necropsy and histopathological findings as well as other toxic effects at the target level, a detailed examination for each single effect is required, probably taking into account the detailed description in the effect additional. This exceeds the feasibility for this evaluation. Body weight changes and developmental landmarks were evaluated at the postnatal stage. Body weight changes of adults were evaluated as main marker for general toxicity Organ toxicity was exemplarily analysed by organ weight changes (Table 8). Overall, the scope of examination is, except for developmental landmarks, more comprehensive in adults. The percentages for body weight alterations at the postnatal and adult stage remain hardly unchanged when the scope of examination is taken into account, reflecting that body weight is monitored in most of the studies (Table 8). In contrast to body weight changes other targets gain relevance because they are not evaluated in each study. Organ weights of liver and kidney represent the most frequently affected targets in adults with the liver even exceeding

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Table 7 Comparison of targets determining the F1-generation LOEL in multi-generation studies at the different developmental stages. Target category

General toxicity

General organ

Male reproductive target Female reproductive target Fertility Endocrine system

Offspring development

Target

Body weight Clinical symptoms Clinical chemistry Mortality Behaviour Immune system Reflex response/reflex ontogenesis Liver Kidney Brain Thymus Spleen Lung Bone Testes Prostate Epididymis Seminal vesicle Ovary Sperm parameter Fertility* Litter size Endocrine system Adrenal gland Thyroid gland Pituitary gland Anogenital distance Vaginal opening Retention of nipples/areolae Testes descent/ectopic testes Preputial separation Eye opening Pinna detachment

Pct. of all studies (N = 169) Adult

Postnatal

Prenatal

18.9 15.4 5.3 3.6 2.4 1.8 1.2 19.5 11.2 3.0 2.4 1.8 1.8 0 8.3 5.3 4.1 3.6 3.6 5.3 3.0 n.a. 6.5 3.0 2.4 1.8 0.6 n.a. 1.8 1.2 n.a. n.a. n.a.

27.8 0.6 3.6 4.1 1.8 0.6 3.0 0 1.2 2.4 1.8 1.2 0 0.6 1.2 1.2 0.6 0.6 0.6 n.a. n.a. 0 2.4 0.6 0.6 0 8.3 4.7 3.6 1.8 1.8 1.8 1.8

7.1 n.a. n.a. 4.1 n.a. n.a. n.a. 0 0 0 0 0 0 3.0 0.6 0.6 0 n.a. 0 n.a. n.a. 4.7 n.a. n.a. 0 n.a. 0 n.a. n.a. 0 n.a. n.a. n.a.

Targets are presented when a percentage of more than 1.5% is achieved in at least one developmental stage. n.a. = target not applicable at this stage. * The target fertility compromises data on mating and fertility indices and number of pregnant females.

the percentage of body weight changes. Sensitive reproductive parameters are organ weights of prostate, seminal vesicles and ovaries as well as altered sperm parameters. The % affected for these targets is nearly doubled in adults, if one relates these effects to the number of studies where these parameters were in fact investigated (Table 8). The frequency of developmental landmarks and hormone regulated parameters also strongly increases. With about one third of the studies changes of anogenital distance and an observed retention of nipples/areolae even exceed the value for body weight change at the postnatal stage. Testicular descent, vaginal opening and pinna detachment (as parameters of general offspring development as well as endocrine regulation) are also highly affected. The persistence of the endocrine regulated parameters until the adult stage was also investigated in few studies showing comparably high percentages for retention of nipples/areolae and ectopic testes.

related structures. In addition well known developmental or reproductive toxicants (e.g. pentachlorophenol, tertiary amyl methyl ether, cyclosporine A) were also indicated as substances with a related MoA. Although this classification might not be exhaustive, Table 9 shows a clear influence of the MoA on the scope of examination: 56 to 100% of the substances tested for one of the developmental landmarks are endocrine active or reproductive toxic substances. From 78% to 100% of the substances for which an effect was observed, the potential MoA is known. But it has to be emphasised that in contraposition the appearance of certain target parameters should not be used to conclude a mode of action, as e.g. delays in vaginal opening or preputial separation may occur as consequences of developmental toxicity (correlating with decreased pup body weight) or as consequences of endocrine disruption. 3.8. Effects on fertility of F1

3.7. Chemical bias on developmental landmarks for sexual maturation To test the hypothesis that certain developmental landmarks, especially on sexual maturation, are investigated not generally but preferably in compounds with a known or suspected hormonal mode of action (MoA), all compounds for which the respective landmarks were assessed, were distinguished according to their MoA. The compounds with potentially endocrine MoA comprise hormones (e.g. thyroxine), experimental hormones (e.g. testosterone propionate, 17-beta-estradiol), hormonally active pharmaceuticals (e.g. tamoxifen, finasteride), known endocrine disruptors (e.g. vinclozolin, flutamide, butylbenzyl phthalate) and chemicals with

Comparing NOEL ratios of FeDTex studies, the F2 generation contributes, compared to the F1, only in exceptional cases to the study NOEL in multi-generation reproduction studies (Table 4). Besides the effects on the developing F2 offspring encompassing survival and development, the mating of F1 may also result in data on impaired reproductive capacity of the F1 generation encompassing mating, fertility and gestation index, gestation length, signs of dystocia. This would increase the information on F1 response but on costs of generating a whole new generation of animals. It is thus analysed, if the data assessed so far in the F0 and F1 generation are sufficient to provide indication on fertility impairment without mating of F1.

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Table 8 Comparison of targets determining the F1-generation LOEL in multi-generation studies (N = 169) taking the scope of examination into account. Target category

General toxicity

General organ weight

Male reproductive organ weight Female reproductive organ weight Fertility Endocrine organ weight

Offspring development

Target

Body weight Mortality Liver Kidney Thymus Brain Lung Spleen Prostate Seminal vesicle Testes Epididymis ovary uterus sperm parameter Thyroid gland Adrenal gland Pituitary gland Anogenital distance Retention of nipples/areolae Testes descent Vaginal opening Pinna detachment Eye opening Preputial separation

Postnatal

Adult No. affected

No. examined

Pct. affected/ examined

Pct. affected/ all studies (169)

No. affected

No. examined

Pct. affected/ examined

Pct. affected/ all studies (169)

32 6 26 15 3 6 1 2 9 6 6 5 6 2 9 3 4 3 1

154 169 98 86 28 60 14 52 80 76 106 90 79 58 73 26 59 48 6

21 4 27 17 11 10 7 4 11 8 6 6 8 3 12 12 7 6 17

19 4 15 9 2 4 1 1 5 4 4 3 4 1 5 2 2 2 1

47 7 0 0 2 4 0 2 2 1 2 1 1 0 n.a. 0 1 0 14

165 169 18 15 34 36 1 36 10 12 24 19 24 20 n.a. 4 8 6 40

28 4 0 0 6 11 0 6 20 8 8 5 4 0 n.a. 0 13 0 35

28 4 0 0 1 2 0 1 1 1 1 1 1 0 n.a. 0 1 0 8

3

6

50

2

6

19

32

4

2 n.a. n.a. n.a. n.a.

3 n.a. n.a. n.a. n.a.

67 n.a. n.a. n.a. n.a.

1 n.a. n.a. n.a. n.a.

3 8 3 3 3

20 73 31 37 67

15 11 10 8 4

2 5 2 2 2

Targets are presented when a percentage affected/examined of more than 1.5% is achieved in at least one developmental stage. n.a.= target not applicable at this stage.

Therefore, we compare effects on reproductive organs and effects on fertility in the F0 and F1 generation. Our data show that the mating of F1 does not add relevant information. Effects are observed only in 5 multi-generation studies at the study LOEL level. Analysis of these 5 studies (Table 10) showed that in 3 studies fertility was decreased also in the F0 generation at the same dose. In one study a decreased male fertility index was observed for the second F2 litter (F2b) only. This effect was neither observed while producing the F2a offspring nor at a higher dose and was therefore considered incidental. The last study showed a higher sensitivity of the F1 for decreased fertility compared to F0 by a factor of 2. However, at the same dose level the weight of the reproductive organs was decreased, therefore also in this case effects on fertility are adequately detected without producing an F2. 3.9. Effects observed either in F1 or F2 offspring only Following the quantitative analysis of offspring responsiveness using the NOEL/LOEL approach and analysis of the impact

of the scope of examination on effect observation, the qualitative aspect of effect occurrence in F1 and F2 offspring was analysed irrespective of dose levels. In 43 out of 138 studies effects were solely observed in F1 but not in F2, while they are F2-exclusive in 29 studies (Table 11). In the majority of the 29 studies, the F2 effects were body weight changes or organ weight alterations, decreased litter size, increased offspring mortality, or effects on developmental landmarks (a detailed analysis of the F2-exclusive effects is provided in Supplementary 3). In 25 out of the 29 studies, the existing F1 data (toxicity in pre-weaning F1 pups, adverse effects on developmental landmarks or sexual maturation) or an impaired F0 fertility would have triggered F1 mating in the EOGRTS (extended-one-generation-reproductive-toxicity-study). In 2 of the remaining 4 studies no relevant findings would have been missed as the F2 findings were limited to a decreased postnatal body weight or altered organ weights were observed only in the presence of parental toxicity. This leaves 2 critical studies. In one case postnatal mortality is increased in F2, but the effect is of doubtful relevance as mortality was not increased in the high dose group.

Table 9 Developmental landmarks for sexual maturation assessed in F1 in multi-generation studies: influence of the testing chemical and its mode of action (MoA) on the scope of examination. Target

Vaginal opening Anogenital distance Nipple retention Preputial separation Testicular descent

Pct. of chemicals with

No. of chemicals tested

64 36 16 59 18

related MoA

effect observed

observed effect attributable to related MoA

56 75 100 58 72

14 36 44 7 17

78 92 100 100 100

252

F. Schulz et al. / Toxicology Letters 226 (2014) 245–255

Table 10 Analysis of effects in reproductive organs in studies with decreased fertility in F1 at the study LOEL. Study no.

Dose groups

Effects on reproductive organs in F0*

1

2.5 mg/kg bw 10 mg/kg bw 40 mg/kg bw

No effects described

2

3

4

Testes weight ↓; degenerative changes in testes, epididymis, and seminal vesicles; Impaired sperm parameters

50 ppm

Testicular spermatic granulomas ↑ Degenerative changes in testes; impaired sperm parameters

*

Fertility ↓

F0 = F1

No effects described

F0 = F1 No. of litters ↓ (not significant)

No effects described

Incidental Male fertility index ↓ in 2nd mating of F1, effect not seen at higher dose or 1st mating of F1

450 ppm 1000 ppm

2000 ppm 4000 ppm

Fertility ↓

No effects described No. of litters ↓; Males proven less fertile Testes weight ↓; atrophy of semiferous tubules ↑; severe bilateral testicular degeneration of Sertoli’s cells and sperma-togonia ↑; No. of primary spermatocytes and/or secondary spermatocytes ↓; epididymal granulomas/sloughed spherical cells in lumen ↑; epididymal sperm count↓ No effects described

Evaluation

Precoital interval ↑

150 ppm

5

Effects on Fertility of F1*

F0 = F1

Fertility ↓

150 ppm 475 ppm

1500 ppm

Effects on reproductive organs in F1*

Fertility ↓

1000 ppm

2000 ppm 4000 ppm

Effects on fertility of F0*

Precoital interval ↑ Oestrus cycle length ↑

Weight of epididymis, prostate, seminal vesicles ↓

Mating and fertility index ↓

F0 = F1

Fertility ↓

Effect LOELs; ↑ increased; ↓ decreased.

The remaining study had already been identified in Section 3.3. It showed structural abnormalities in bone and kidneys pointing to growth retardation as critical effect. 3.10. Correlation between body weight effects and offspring developmental parameters It was next analysed whether delayed maturation is associated with or a consequence of reduced body weight (Table 12). Among 452 studies with reported F1-effects the majority (358 studies) provided prenatal/postnatal body weight or maturational effect data. In 157 studies only effects in offspring body weight without effects on other maturational data were reported, while 49 studies displayed a delay in maturation (decreased skeletal ossification at Caesarean section, delayed achievement of developmental

Table 11 Evaluation of effect occurrence in the F1 and F2 offspring generation. Pct. of studies (N = 138)

Studies with effects Studies with effects observed only in one generation Studies with effects exclusive for one generation (not covered by similar effects or in different developmental stage of the other generation)

F1

F2

95 91

75 35

31

21

landmarks, reflex ontogenesis or sexual maturation) without effects on pup body weight. Among the remaining studies, 127 studies displayed a clear relationship of decreased offspring body weight with a maturational delay. In 4 studies an increased body weight was correlated with accelerated maturation and further 4 studies had a clear reverse correlation. One study was not included Table 12 Analysis of the co-occurrence of offspring body weight alteration and maturational delay.

Effects in F1 Prenatal/postnatal body weight affected and/or alteration in maturation Prenatal/postnatal body weight affected only Delay in maturation only Prenatal/postnatal body weight decreased and delay in maturation Prenatal/postnatal body weight increased and acceleration in maturation Reverse correlation of body weight alteration and maturational effects Sex-dependent endocrine effects from known EDCs potentially superimposed a possible correlation

Number of studies

Pct. of studies (N = 535)

452 358

84 67

157

29

49 127

9 24

4

Sensitivity of different generations and developmental stages in studies on reproductive toxicity.

Numerous studies on reproductive toxicity are expected to be necessary under the EU program on Registration, Evaluation, Authorisation and Restriction...
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