Aop: 212


Each AOP should be given a descriptive title that takes the form “MIE leading to AO”. For example, “Aromatase inhibition [MIE] leading to reproductive dysfunction [AO]” or “Thyroperoxidase inhibition [MIE] leading to decreased cognitive function [AO]”. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Histone deacetylase inhibition leading to testicular atrophy

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
Histone deacetylase inhibition leading to testicular atrophy

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help


List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

Shihori Tanabe, Akihiko Hirose, Takashi Yamada

Division of Risk Assessment, Biological Safety Research Center, National Institute of Health Sciences

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Shihori Tanabe   (email point of contact)


List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Shihori Tanabe


The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
Author status OECD status OECD project SAAOP status
Open for citation & comment EAGMST Under Review 1.52 Included in OECD Work Plan
This AOP was last modified on February 16, 2021 00:55
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

Revision dates for related pages

Page Revision Date/Time
Histone deacetylase inhibition February 10, 2020 03:09
Histone acetylation, increase February 10, 2020 03:15
Cell cycle, disrupted February 10, 2020 03:18
Apoptosis February 10, 2020 03:23
Testicular atrophy February 10, 2020 03:38
Spermatocyte depletion February 10, 2020 03:31
Histone deacetylase inhibition leads to Histone acetylation, increase February 10, 2020 03:50
Histone acetylation, increase leads to Cell cycle, disrupted February 10, 2020 04:04
Cell cycle, disrupted leads to Apoptosis February 10, 2020 04:42
Histone deacetylase inhibition leads to Cell cycle, disrupted February 10, 2020 04:27
Apoptosis leads to Spermatocyte depletion February 10, 2020 04:16
Histone deacetylase inhibition leads to Apoptosis February 10, 2020 04:31
Spermatocyte depletion leads to Testicular atrophy February 10, 2020 04:22
Histone deacetylase inhibition leads to Spermatocyte depletion February 10, 2020 04:34
Histone deacetylase inhibition leads to Testicular atrophy February 10, 2020 04:40
Methoxyacetic acid January 21, 2018 20:38
Butyrate January 21, 2018 20:39
Trichostatin A January 21, 2018 20:39
Valproate November 29, 2016 18:42


In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

Testicular toxicity is of interest for human health risk assessment especially in terms of reproductive and developmental toxicity, however, the testicular toxicity has not fully elucidated. Histone deacetylase inhibitors (HDIs) are approved as anti-cancer drugs, since HDIs have apoptotic effect in cancer cells. HDIs includes the short chain fatty acids, hydroxamic acids, benzamides and epoxides. The intracellular mechanisms of induction of the spermatocyte apoptosis by HDIs are suggested as histone deacetylase (HDAC) inhibition as MIE, histone acetylation increase, disrupted cell cycle, apoptosis, and spermatocyte depletion as KEs. Adverse outcome has been defined as testicular atrophy. The HDIs inhibit deacetylation of the histone, leading to the increase in histone acetylation. The apoptosis induced by disrupted cell cycle leads to spermatocyte depletion and testis atrophy. This AOP may be one of the pathways induced by HDIs, which suggests the pathway networks of protein hyperacetylations.

Abbreviation: AOP: adverse outcome pathway, HDAC: histone deacetylase, HDI: HDAC inhibitor, KE: key event, MIE: molecular initiating event

Background (optional)

This optional subsection should be used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development. The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

Summary of the AOP

This section is for information that describes the overall AOP. The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help


Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
Sequence Type Event ID Title Short name
1 MIE 1502 Histone deacetylase inhibition Histone deacetylase inhibition
2 KE 1503 Histone acetylation, increase Histone acetylation, increase
3 KE 1505 Cell cycle, disrupted Cell cycle, disrupted
4 KE 1262 Apoptosis Apoptosis
5 KE 1515 Spermatocyte depletion Spermatocyte depletion
6 AO 1506 Testicular atrophy Testicular atrophy

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarises all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP. Each table entry acts as a link to the individual KER description page.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help


The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help
Name Evidence Term
Methoxyacetic acid High
Butyrate High
Trichostatin A High
Valproate Moderate

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
Adult, reproductively mature High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
human Homo sapiens Moderate NCBI
mouse Mus musculus Moderate NCBI
rat Rattus norvegicus High NCBI

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence
Male High

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help
Attached file: Overallassessmentaop212rev 2 10 20

1. Support for Biological Plausibility of KERs

MIE => KE1: Histone deacetylase inhibition leads to histone acetylation increase

Biological Plausibility of the MIE => KE1 is high. Rationale: Upon the inhibition of HDAC by HDIs, the acetylation of lysine in histone remains and it leads to transcriptional activation or repression, changes in DNA replication and DNA damage repair. Activity of histone acetyltransferase (HAT) in testis nuclear protein was increased with MAA addition [Wade et al., 2008].

KE1 => KE2: Histone acetylation, increase leads to cell cycle, disrupted

Biological Plausibility of the KE1 => KE2 is moderate. Rationale: Gene transcription is regulated by histone acetylation [Struhl, 1998]. Acetylation of histones neutralizes the positive charge of the histones. Thus, less compacted DNA can be bound more easily by transcription factors and transcribed. In the models proposed for the relationship between histone acetylation and transcription, histone acetylation can be untargeted and occur at both promoter and nonpromoter regions, targeted generally to promoter regions, or targeted to specific promoters by gene-specific activator proteins [Richon et al., 2000; Struhl, 1998].

KE2 => KE3: Cell cycle, disrupted leads to apoptosis

Biological Plausibility of the KE2 => KE3 is moderate. Rationale: Prolonged cell cycle arrest will lead to either senescence or apoptosis. Especially for fast dividing and still differentiating cells, such an arrest will most certainly induce apoptosis as the normal cellular program cannot be followed.

KE3 => KE4: Apoptosis leads to spermatocyte depletion

Biological Plausibility of the KE3 => KE4 is moderate. Rationale: During development and in tissue homeostasis, apoptosis is needed to control organ size. If apoptosis is induced at a higher rate, one can assume it leading to atrophy of the target organ. Especially when target organ / target cells are fast replicating, abnormal levels of apoptosis will lead to depletion.

KE4 => AO: Spermatocyte depletion leads to testicular atrophy

Biological Plausibility of the KE4 => AO is moderate. Rationale: Spermatocyte depletion is one of the main characteristics of testicular atrophy.

2. Support for essentiality of KEs

KE2: Cell cycle, disrupted

Essentiality of the KE2 is moderate. Rationale for Essentiality of KEs in the AOP: HDAC1-defecient embryonic stem cells showed reduced proliferation rates, which correlates with decreased cyclin-associated kinase activities and elevated levels of the cyclin-dependent kinase inhibitor 1A, a cell cycle regulator p21 [Lagger et al., 2002]. Loss of HDAC1 leads to significantly reduced overall deacetylase activity, hyperacetylation of a subset of histones H3 and H4 [Lagger et al., 2002].

3. Empirical support for KERs

MIE => KE1: Histone deacetylase inhibition leads to histone acetylation, increase

Empirical Support of the MIE => KE1 is high. Rationale: HDAC inhibitors increase histone acetylation in brain [Schroeder et al., 2013]. The major empirical evidence came from the incubation of cell culture cells with small molecule compounds that inhibit HDAC enzymes followed by western blots or acid urea gel analysis. The first evidence was shown by Riggs et al. who showed that incubation of HeLa cells with n-butyrate leads to an accumulation of acetylated histone proteins [Riggs et al., 1977]. Later, it was shown that n-butyrate specifically increases the acetylation of histone by the incorporation of radioactive [3H]acetate and analysis of the histones on acid urea gels that allow the detection of acetylated histones [Cousens et al., 1979]. TSA was shown to be an HDAC inhibitor by acid urea gel analysis in 1990 [Yoshida et al., 1990] and good evidence for VPA as an HDAC inhibitor in vitro and in vivo was shown using acetyl-specific antibodies and western blot [Gottlicher et al., 2001].

KE1 => KE2: Histone acetylation, increase leads to cell cycle, disrupted

Empirical Support of the KE1 => KE2 is moderate. Rationale: Increase in histone acetylation by HDAC inhibition induces the cell cycle regulator expression and inhibits progression through the cell cycle. Histone acetylation regulates the gene transcriptional mechanism [Struhl, 1998]. Acetylation of histones promotes the RNA polymerase reaction [Allfrey et al., 1964; Pogo et al., 1966]. Since several results supported a model in which increased histone acetylation is targeted to specific gene and occurs throughout the entire genome, not just the promoter regions, histone acetylation may lead to gene transcription of the cell cycle regulator [Richon et al., 2000].

KE2 => KE3: Cell cycle, disrupted leads to apoptosis

Empirical Support of the KE2 => KE3 is moderate. Rationale: Cell cycle arrest such as G1 arrest and G1/S arrest are observed in apoptosis [Li et al., 2012; Dong et al., 2010]. microRNA-1 and microRNA-206 represses CCND2, while microRNA-29 represses CCND2 and induces G1 arrest and apoptosis in rhabdomyosarcoma [Li et al., 2012].

KE3 => KE4: Apoptosis leads to spermatocyte depletion

Empirical Support of the KE3 => KE4 is high. Rationale: MicroRNA-21 regulates the spermatogonial stem cell homeostasis, in which suppression of microRNA-21 with anti-miR-21 oligonucleotides led to apoptosis of spermatogonial stem cell-enriched germ cell cultures and the decrease in the number of spermatogonial stem cells [Niu et al., 2011].

KE4 => AO: Spermatocyte depletion leads to testicular atrophy

Empirical Support of the KE4 => AO is high. Rationale: The testicular atrophy seen in 2-methoxyethanol (2-ME), or its major metabolite MAA, treated rats in vivo and in human, and rat in vitro culture was associated with spermatocyte depletion [Beattie et al., 1984].

Domain of Applicability

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

The AOP is applicable to the reproductively mature males in rats, mice and humans. The administration route or doses of HDAC inhibitors may affect the intensity of the toxicity.

Essentiality of the Key Events

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence.The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help
Key Event Direct/Indirect Evidence
MIE: Histone deacetylase inhibition HDAC inhibition induced testicular toxicity including testis atrophy [Miller et al., 1982]. HDAC inhibition in cell culture resulted in the testicular toxicity including germ cell apoptosis and cell morphology change [Li et al., 1996].
KE1: Histone acetylation, increase The HDAC inhibition induced cell death in spermatocytes in both rat and human seminiferous tubules [Li et al., 1996].
KE2: Cell cycle, disrupted In HDAC1-/- fibroblast lines, increase in the amount of cells in G1 phase and decrease in the amount of cells in S phase were observed, which indicates the importance of HDAC inhibition in cell cycle regulation [Zupkovitz et al., 2010].
KE3: Apoptosis HDAC inhibition leads to cell death through the apoptotic pathways [Falkenberg et al., 2014].
KE4: Spermatocyte depletion The HDAC inhibition induced cell death in spermatocytes in both rat and human seminiferous tubules [Li et al., 1996]. The HDAC inhibitor treatment resulted in degeneration in spermatocytes in rat seminiferous tubules [Li et al., 1996]. The HDAC inhibition induced the germ cell apoptosis in human testicular tissues [Li et al., 1996].

Evidence Assessment

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

Biological plausibility, coherence, and consistency of the experimental evidence

The available data supporting the AOP are logic, coherent and consistent with established biological knowledge, whereas there are possibilities for alternative pathways.


Alternative mechanism(s) that logically present themselves and the extent to which they may distract from the postulated AOP

There are some other important apoptotic pathways that are involved in cell death, as well as other important spermatocyte signaling or mechanism influences testicular toxicity.


p53 pathway

The study in which in vivo administration of trichostatin A (TSA), a HDI, in mice resulted in male meiosis impairment showed the involvement of p53-noxa-caspase-3 apoptotic pathway in TSA-induced spermatocyte apoptosis [Fenic et al., 2008]. Other study showed that MAA induced up-regulation of p21 expression is mediated through histone hyperacetylation and independent of p53/p63/p73 [Parajuli et al., 2014].


      NF-kappaB pathway

The present AOP focuses on p21 pathway leading to apoptosis, however, the alternative pathway such as NF-kappaB signaling pathways may be involved in apoptosis of spermatocytes [Wang et al., 2017].


Communication with Sertoli cells

The present AOP focuses on testicular atrophy by HDAC inhibition-induced apoptosis in spermatocytes, however, the signaling in Setoli cells may be involved in testicular atrophy. Sertoli cell secretes GDNF, FGF2, CXCL12 or Ccl9 molecules, which results in the activation of RET, FGFR, CXCR4 or CCR1 signaling in spermatogonial stem cells, respectively [Chen and Liu, 2015].


Decrease in deoxynucleotide pool by MAA

MAA induces decrease in deoxynucleotide pool, resulting apoptosis, which may be an alternative pathway other than p21-mediated pathway [Yamazoe et al., 2015]. Inhibition of 5,10-CH2-THF production by MAA may decreases deoxynucleotide pool in spermatocytes [Yamazoe et al., 2015].

Spermatocyte depletion by necrosis

Spermatocyte may be decreased by necrosis. Cell death mechanism other than apoptosis, such as necrosis, may be considered for spermatocyte depletion.

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

Concordance of dose-response relationships

This is a quantitative description on dose-response relationships from MIE to AOP. But some KE relationships individually are not fully supported with dose-response relationships, while there is empirical evidence to support that a change in KEup leads to an appropriate change in the respective KEdown.


Temporal concordance among the key events and adverse outcome

Temporal concordance between MIE and AOP has been described with in vivo experimental data. Empirical evidences show temporal concordance between MIE and the individual KEs, however, the temporal concordance among the individual KEs and AO is not fully elucidated.

Strength, consistency, and specificity of association of adverse outcome and initiating event

The scientific evidence on the linkage between MIE and AO has been described.

The quantitative understanding of the AOP in terms of indirect relations between HDAC inhibition and testicular atrophy was examined in in vivo experiments [Foster et al., 1983; Miller et al., 1982].


Considerations for Potential Applications of the AOP (optional)

At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

The present AOP can be used in risk assessment of several types of the molecules including HDAC inhibitors as the anti-cancer drugs, as well as another types of the chemicals, in terms of testicular toxicity. HDAC inhibitors nowadays have been utilized as therapeutics for cancer or neurology disease, and the adverse effects of HDAC inhibitors should be evaluated. This AOP elucidating the pathway from HDAC inhibition to testicular atrophy may provides important insights for potential toxicity of HDAC inhibitors. It also provides a basis for the HDAC inhibition-induced epigenetic alteration and cell death. HDAC inhibitors such as rocilinostat are clinically evaluated as anti-cancer drugs in clinical trial.


List the bibliographic references to original papers, books or other documents used to support the AOP. More help

Allfrey, V. et al. (1964), "Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis", Proc Natl Acad Sci 51:786-794

Beattie, P.J. et al. (1984), "The effect of 2-methoxyethanol and methoxyacetic acid on Sertoli cell lactate production and protein synthesis in vitro", Toxicol Appl Pharmacol 76:56-61

Chen, S. and Liu, Y. (2015), "Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling", Reproduction 149:R159-R167

Cousens, L.S., et al. (1979), "Different accessibilities in chromatin to histone acetylase", J Biol Chem 254:1716-1723

Dong, Q. et al. (2010), "microRNA let-7a inhibits proliferation of human prostate cancer cells in vitro and in vivo by targeting E2F2 and CCND2", PLoS One 5:e10147

Fenic, I. et al. (2008), "In vivo application of histone deacetylase inhibitor trichostatin-A impairs murine male meiosis", J Andro 29:172-185

Foster, P.M. et al. (1983), "Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rat", Toxicol Appl Pharmacol 69:385-39

Gottlicher, M. et al. (2001), "Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells", EMBO J 20:6969-6978

Lagger, G. et al. (2002), "Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression", EMBO J 21:2672-2681                                       

Li, L. et al. (2012), "Downregulation of microRNAs miR-1, -206 and -29 stabilizes PAX3 and CCND2 expression in rhabdomyosarcoma", Lab Invest 92:571-583  

Li, L.H. et al. (1996), "2-Methoxyacetic acid (MAA)-induced spermatocyte apoptosis in human and rat testes: an in vitro comparison", J Androl 17:538-549

Miller, R.R. et al. (1982), "Toxicity of methoxyacetic acid in rats", Fundam Appl Toxicol 2:158-160

Niu, Z. et al. (2011), "microRNA-21 regulates the self-renewal of mouse spermatogonial stem cells", Proc Natl Acad Sci 108:12740-12745

Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-312

Pogo, B. et al. (1966), "RNA synthesis and histone acetylation during the course of gene activation in lymphocytes", Proc Natl Acad Sci 55:805-812

Richon, V.M. et al. (2000), "Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation", Proc Natl Acad Sci 97:10014-10019        

Riggs, M.G. et al. (1977), "N-butyrate causes histone modification in HeLa and friend erythroleukaemia cells", Nature 268:462-464

Schroeder, F.A. et al. (2013), "A selective HDAC 1/2 inhibitor modulates chromatin and gene expression in brain and alters mouse behavior in two mood-related tests", PLoS One 8:e71323

Struhl, K. (1998), "Histone acetylation and transcriptional regulatory mechanisms", Gene Dev 12:599-606      

Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831

Wang, C. et al. (2017), "CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways", Oncotarget 8:3132-3143

Yamazoe, Y. et al. (2015), "Embryo- and testicular-toxicities of methoxyacetate and the related: a review on possible roles of one-carbon transfer and histone modification", Food Safety 3:92-107

Yoshida, M. et al. (1990), "Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro trichostatin A", J Biol Chem 265:17174-17179

Zupkovitz, G. et al. (2010), "The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation", Mol Cell Biol 30:1171-1181