Workshop Report 27

Review of the terms of reference and hypotheses

The group provided constructive challenge to the draft hypotheses, resulting in extensive revision.

Original Hypothesis 1

“Endocrine active chemicals do not have a threshold below which adverse effects are not seen and should be regarded and tested in a different way to chemicals acting through other modes of action.”

Group consensus

As written the statement was not supported by the group as a hypothesis and was considered essentially meaningless.

The hypothesis as written cannot be tested or demonstrated by observational means for chemicals acting through any mode of action including endocrine. A key obstacle is the statistical power needed to detect low effect sizes that may be assumed at low threshold dose levels.


The hypothesis, derived from studies with very potent oestrogenic chemicals, may not be relevant for low potency oestrogens or chemicals acting via other MoAs (e.g. prostaglandin synthesis inhibitors). The hypothesis needs to be re-formulated and focussed on specific areas for investigation:

• Questions need to be posed on a case-by-case basis.
• These should come from an understanding of the MoA and key events for a specific chemical or class.
• The dose response relationship for key events in a MoA or in an Adverse Outcome Pathway (AOP) will need to be understood to enable it to be investigated as will any dose response relationships for effects that may be considered adverse.
• Adverse outcome needs to be integrated using an understanding of relationships between the ultimate adverse effect, key events, the site of possibly multiple actions and the exposure, and such integration is likely to require a longer term initiative.

Based on the above discussions and proposals a revised hypothesis was drafted and agreed:

“The lack of a threshold means that the dose response relationships established for endocrine disrupters will show adverse effects over a very wide range of dose levels.”

Original Hypothesis 2

“At low dose / exposure levels, endocrine active chemicals exhibit non-monotonic dose responses.”

Group consensus

As written, the statement is not a compelling hypothesis for valid scientific investigation. However if it were to be made more specific then there is some scope for empirical investigation.


The hypothesis needs to be re-formulated, made more specific and to explicitly include the concept of adversity:

• Focus on addressing the question for chemicals where there is sufficient understanding of the MoA that leads to adverse effects. Consequently, for completeness, this work could involve studying several different MoAs that are presently relevant in the field of endocrine disruption.
• Focus on chemicals where there are claims about the existence of NMDRs.

• Model established key events and use selected in vivo approaches. Well defined in vitro studies using dose-response for markers related to adversity can also complement the in vivo approach.
• Ensure the MoA understanding is able to distinguish adverse events from homeostatic effects.
• Ensure the approach is statistically robust; understand and control sources of variation.
• Understand the dose range and ensure a sufficient number of dose levels is used to provide refined knowledge in line with the hypothesis.
• Ensure that the level of change constituting a real change in direction of the dose response is understood and agreed.

Based on the above discussions and proposals a revised hypothesis was drafted and agreed:

“Non-monotonic responses in dose response relationships for endocrine disrupters mean that it is not possible to anticipate adverse events using traditional approaches.”

Original Hypothesis 3

“When mixed at low doses, endocrine active chemicals produce effects greater than those which may be predicted from simple dose addition.”

Group consensus

This is not a priority area in the first instance and any further work should take into account and learn from the numerous extensive reviews and position papers already in the public domain – EU State of the Art Report (Kortenkamp et al, 2011; Boobis et al, 2011; EU, 2011; ECETOC, 2012).


• There is some potential to investigate the hypothesis but activities should be focussed in areas where synergy may be predicted from existing knowledge of MoA.
• Need to agree and use definition and understanding of low dose.
• Modelling and prediction should be based on MoA data.
• Need a clear agreement of what level of change / effect would be considered greater than that predicted by dose addition alone.

Based on the above discussions and proposals a revised hypothesis was drafted and agreed but it was not considered a priority area for further work at this time.

“MoA understanding can be used to predict ‘synergy’ when chemicals are mixed and when tested these mixtures will show ‘synergy’.”

In summary:

• The group provided clear guidance on how the hypotheses and terms of reference should be revised.
• Further consideration of thresholds and non-monotonic dose responses were clearly identified and agreed as the priority areas. Although, not identified as a top priority, Mixture effects should be addressed at a later stage in the research programme.

• The group agreed that traditional effect-led approaches to further study low dose and likely low effect responses was scientifically inappropriate, technically highly challenging with respect to experimental design and the number of experimental subjects required to achieve a robust outcome. For these reasons the group unanimously supported a MoA/AOP led approach, with the focus on key events, their quantitative description and dose response characteristics, as best way to understand threshold and low dose effects.