FIFRA SCIENTIFIC ADVISORY

PANEL MEETING

JULY 30 – AUGUST 2, 2013

Weight-of-Evidence: Evaluating Results of EDSP 

Tier 1 Screening

CHARGE QUESTIONS
Background

 The current case study analyses are primarily focused on the 

interpretative process for determining whether or not there is a chemical 

interaction with estrogen, androgen, or thyroid (E, A, or T) signaling 

pathways. An important aspect of addressing this question includes 

characterizing at what level of biological organization (e.g., molecular, 

cellular, tissue/organ, organism) that interaction is expressed, the 

robustness of the data showing an interaction in terms of the extent of 

complementarity and redundancy across multiple lines of evidence, and 

describing under what conditions (e.g., in an organism with an intact 

hypothalamic-pituitary-gonadal axis (HPG), in the absence of overt 

toxicity, at what doses, duration and route of exposure, etc.) the 

chemical interacts with and perturbs the endocrine system.
Background continued...

 The charge questions are organized around the fundamental steps 

in the WoE guidance document in addressing whether or not a 

chemical interacts with the E, A, or T signaling pathways: (i) 

assembling and evaluating the scientific quality of the individual 

studies, and (ii) formulating hypotheses and integrating data at 

different levels of biological organization, which includes 

characterizing the extent and nature of the complementarity and 

redundancy in responses. In the context of these steps in the WoE

guidance document, cross-cutting as well as case study-specific 

questions are provided, consistent with the rationale for selecting 

the example chemicals for this review.
Charge 1

 As described in the 2011 EDSP WoE guidance 

document, in assembling and evaluating the 

quality of scientific information to support a 

determination of a chemical’s potential to interact 

with E, A, or T signaling pathways, it is important 

to ensure data soundness, applicability, utility, 

clarity and completeness.
Charge 1.1

 Please comment on whether the agency has 

transparently described the conduct and 

results of the individual Tier 1 studies and the 

OSRI for each of the case studies, and 

specifically whether the level of detail is 

sufficient to ensure that a study is reliable for 

determining the potential to interact with E, A, 

or T signaling pathways and the rationale for 

the preliminary study conclusion.
Charge 1.2

 For each of the case studies, please 

comment on whether the performance criteria 

are clearly stated for the Tier 1 assays and, 

when results were not within the boundaries 

of the performance criteria, whether EPA has 

clearly expressed why the data are still 

considered reliable.
Charge 1.3

 The test guidelines for Tier 1 assays recommend that the organism is challenged 

by attaining sufficiently high treatment doses/concentrations. Difficult to test 

substances may be encountered in Tier 1 screening. Chemical S is an illustration 

of this situation. In the case of Chemical S, consistent exposure was not achieved 

in the Amphibian Metamorphosis assay (AMA) due to the physical-chemical 

characteristics of the test substance. The compound has low solubility and is highly 

lipophilic (high Kow) and prone to sorbing to surfaces (high Koc). Due largely to 

these properties, the contributing laboratory performing the AMA with Chemical S 

did not achieve a concentration level high enough to produce a response indicative 

of a maximum tolerated dose. Nonetheless, the agency concluded that the data 

were still useful in the WoE analysis. This determination was based on the 

agency’s understanding that while measured exposure concentrations were lower 

than the targeted nominal concentrations, exposure was reasonably quantified and 

that it is not likely that the chemical would be any less problematic to test if the 

study were repeated. Further, while higher exposure concentrations could have 

been achieved in the AMA, the FSTRA indicates that these higher concentrations 

likely would have resulted in overt toxicity.

 Please comment on the agency’s conclusion regarding the utility of the AMA 

data for Chemical S to still reliably evaluate its potential endocrine 

interaction in a WoE analysis.
Charge 2

 The WoE guidance decision framework is a hypothesis-based 

approach that promotes the analysis of effects at different levels 

of biological organization (molecular, cellular, tissue/organ, 

organism) using diverse in vitro and in vivo studies to determine 

whether or not a chemical interacts with E, A, or T signaling 

pathways. Pivotal to this approach is the concept of adverse 

outcome pathways (AOP) or modes of action (MoA). All of the 

case studies illustrate different data situations when 

characterizing the level of biological organization and the 

conditions under which potential endocrine interactions are 

expressed (e.g., only in organisms with intact HPG or HPT 

axes, coincident with overt toxicity, etc.) and varying degrees of 

complementarity and redundancy in responses encountered in 

the WoE analysis.
Charge 2.1

 Chemicals do not necessarily act by one adverse outcome pathway 

(AOP). The case studies provide an illustration of competing AOPs 

(endocrine versus non- endocrine pathways or mechanisms of 

pesticidal mode of action that lead to overt toxicity). Although it is 

important to ensure adequate dosing in studies and reach some level of 

toxicity, overt or pronounced toxicity was relatively common at high 

treatment doses/concentrations in the Tier 1 and OSRI studies in these 

case studies. When overt toxicity is observed in the Tier 1 in vivo 

assays, it is an important objective to integrate the understanding of 

overt toxicity in the context of the apical responses associated with 

potential chemical interactions with E, A, or T signaling pathways. 

These case study analyses include a characterization of the treatmentdependent nature and severity of the overt toxicity as well as the 

specificity of the potential endocrine-related responses coincident with 

the overt toxicity. These analyses inform the weight that is placed on 

certain Tier 1 responses in the presence of overt toxicity. The case 

studies provide illustrations of the different situations encountered.
Charge 2.1.a

 Chemical A can result in cholinergic toxicity given that its pesticidal mode of action 

is cholinesterase inhibition. In particular, overt toxicity was observed at high 

concentrations in the FSTRA. Although a number of endocrine responses were 

observed (e.g., decrease in female VTG, fecundity/fertility, GSI, male tubercles) at 

the highest concentration in the FSTRA, there was also pronounced overt toxicity 

that included abnormal behavior and significant body weight reductions consistent 

with cholinergic intoxication. Given the directionality of the FSTRA responses (i.e., 

decreases in the measured endpoints), EPA concluded that the effects found at the 

high concentration in the FSTRA may not necessarily be reflective of an endocrinemediated response, but rather a reflection of a compromised organism with limited 

ability to maintain reproductive function and homeostasis. Although in male fish, 

overt toxicity was not observed at the intermediate concentration, possible 

endocrine responses were limited to two effects that lacked diagnostic specificity 

(i.e., altered GSI and histology).

 Please comment on how the agency has applied its decision logic to 

integrate an understanding of overt toxicity in the context of observed Tier 1 

in vivo responses , and in particular, the agency’s determination not to place 

weight on the FSTRA high concentration responses coincident with overt 

toxicity.
Charge 2.1.b

 The pesticidal mode of action of Chemical S involves the uncoupling of mitochondrial 

oxidative phosphorylation and resulting in the depletion of ATP. Another plausible mode of 

toxic action is related to its irritation properties including irritation that compromises the 

integrity of the gastro-intestinal tract in mammals leading to restricted caloric intake due to 

reduced food consumption. Reflective of these toxic modes of action, observations in the 

Tier 1 studies and OSRI included body weight reductions, behavioral effects, and 

decreased survival. The majority of potential androgen and estrogen-related responses 

(decreases in testosterone, decreases in male and female gonadal weights, delays in VO 

and PPS, decreases in male fertility, an increase in male GSI and VTG) were coincident 

with this overt toxicity. At concentrations where no apparent overt toxicity occurred, there 

were no endocrine related responses in the FSTRA, and responses in female rats were 

limited to a 2 day delay in VO, and for male rats, a decrease in the weights of two 

androgen-dependent tissues. The majority of Tier 1 responses were decreases in the 

measured endpoints, which were largely expressed in the presence of overt toxicity, are 

consistent with a depletion of ATP and restricted caloric intake. Although male VTG was 

increased in fish this is likely an artifact of a single elevated response.

 Please comment on how the agency has applied its decision logic to integrate an 

understanding of overt toxicity in the context of observed Tier 1 in vivo responses, 

and in particular, on the agency’s determination to place less weight on the Tier 1 in 

vivo responses in the presence of overt toxicity.
Charge 2.1.c

 Chemical N is a cyclic unsaturated ketone whose acute mode of toxic action is 

nonpolar narcosis (toxicologically induced and reversible stages of neural disruption, 

i.e. general anesthesia). Unlike the other case study chemicals, there is no pesticidal 

mode of toxic action for N given that it is an inert ingredient. Testing required reaching 

limit doses/concentrations in order to sufficiently challenge the animal. Potential 

androgen responses only occurred in the FSTRA (decrease female VTG, decrease 

fecundity/fertility, altered histology) and in the male pubertal assay (decreases in 

testosterone, decreases in androgen sensitive tissue weights, delays in PPS) near 

limit doses/concentrations (as described in the white paper and test guidelines). 

However, a significant decrease in female VTG was observed at the intermediate 

dose. Observations of overt toxicity (decreased body weights and feeding) were 

reported in the highest treatment group (i.e., near limit concentrations) in the FSTRA, 

but no overt toxicity was reported in the male pubertal assay. Unlike Chemicals A and 

S, the overt toxicity is not as pronounced for Chemical N. The responses in fish and 

rats at the high dose could be due to a compromised metabolic ability and inability to 

reduce chemical load.

 Please comment on the agency’s analysis in characterizing Tier 1 responses 

that are expressed at or near limit doses where some degree of overt toxicity 

occurs, and the extent to which such responses are considered in the WoE

analysis.
Charge 2.1.d

 The case study analyses described above all 

involve situations in which overt toxicity was 

observed coincident with Tier 1 responses.

 Please comment on the agency’s overall 

approach to characterizing Tier 1 responses 

coincident with overt toxicity and 

determining the weight to be given to such 

responses.
Charge 2.2

 In certain case studies, there was a lack of anticipated complementary and 

redundant responses (within an in vivo assay or across assays) at different levels of 

biological organization (molecular, cellular, tissue/organ, and organism) indicative of a 

chemical interaction with an endocrine signaling pathway. The estrogen signaling 

pathway will be used as an illustration. In the case of Chemical N, the mammalian 

assays were negative and responses within the FSTRA did not progress to higher 

level responses (e.g., an effect on VTG did not translate to an effect on gonadaltissue or on fecundity). In the case of Chemical A, the rat uterotrophic and female 

pubertal assays (i.e., an organism with an intact hypothalamic-pituitary-gonadal axis) 

were negative for estrogen-related responses, and although there were some 

responses in the FSTRA in the absence of overt toxicity, they lacked diagnostic 

specificity (e.g., effects on male gonadal tissue or GSI). Given the lack of 

complementarity and redundancy in responses within and across assays, the agency 

considered these situations as insufficient to support a robust conclusion of an 

interaction with endocrine signaling pathways.

 Please comment on the decision logic the agency has used to characterize 

these types of situations where there is a lack of robustness in terms of 

complementarity and redundancy, and the transparency and reasonableness of 

the approach.
Charge 2.3

 In contrast to the situation described in Charge 2.2., Chemical J appears 

to interact with the estrogen signaling pathway in terms of 

complementarity and redundancy across multiple levels of biological 

organization as evidenced through altered steroidogenesis, resulting in 

decreased VTG in female fish which in turn translates to a higher-level 

response (e.g., reduced fecundity) in fish. However, this biological 

continuum was not observed in the Tier 1 rat female pubertal assays and 

the Part 158 mammalian data.

 Please comment on the how the agency has characterized this 

endocrine interaction at different levels of biological organization 

across taxa, and the transparency and reasonableness of the 

conclusions drawn. Please include in your response, comments 

regarding the agency’s conclusion about differences in 

sensitivities between taxa (i.e., fish and rats), regarding chemicals 

that appear to alter steroidogenesis.
Charge 2.4

 Chemical A illustrates a situation where a molecular event has been initiated 

along a pathway via binding to the androgen receptor and by altered 

steroidogenesis, with corroborative evidence from the Hershberger assay. 

However, at a higher level of biological organization, an anti-androgenic response 

is not expressed within the context of the mammalian intact hypothalamicpituitary-gonadal axis (based on the Tier 1 mammalian assays and the 

mammalian in vivo OSRI). In the absence of overt toxicity, there were some 

possible endocrine-related responses in the FSTRA, but they lacked diagnostic 

specificity (e.g., reduced GSI and altered histology). The agency concluded that 

although there is evidence of an endocrine interaction (i.e., the androgen 

signaling pathway) at lower levels of biological organization, clear endocrinedriven responses are not expressed at higher levels of biological organization in 

organisms with an intact HPG-axis, presumably due to compensatory processes.

 Please comment on how the agency has integrated different sources of 

data along a biological continuum to characterize endocrine interactions of 

Chemical A and the transparency and reasonableness of the decision logic.
Charge 2.5

 In some chemical situations, the in vitro Tier 1 data are negative. Nonetheless, 

this does not necessarily detract from a conclusion of a potential endocrine 

interaction in vivo either because a different molecular initiating event (MIE) may 

be occurring than what the in vitro assay evaluates or because an activated 

metabolite may be responsible for the in vivo effects. Chemicals N and S provide 

an illustration of this situation in that the MIE is uncertain due to the negative Tier 

1 in vitro assays. But, there were Tier 1 in vivo responses that are consistent with 

potential interactions with the androgen or estrogen signaling pathways.

 For Chemical N, anti-androgen related responses were observed in the male 

pubertal assay that were complementary within the assay (i.e., decreased in 

testosterone levels that progressed to effects at the organ (tissue weight 

decreases in androgen sensitive tissues) and organism level (delay in PPS). In 

the FSTRA, more limited responses were observed in the absence of overt 

toxicity, i.e., a decrease in female VTG that did not manifest into higher level 

effects. In this case, there is in vivo evidence of an endocrine interaction but 

compared to other case studies (e.g., as Chemical J), the complementarity and 

redundancy in responses are not as robust.
Charge 2.5 continued...

 In the case of Chemical S for the A pathway, in the Hershberger there was a 

decrease in androgen-sensitive tissue weights. In the case of the male pubertal 

assay, there were complementary responses in that a cellular response (i.e., 

decreases in testosterone levels) progressed to effects at the organ (tissue 

weight decreases in androgen sensitive tissues) and organism level (delay in 

PPS). In the FSTRA, there were altered male gonadal weights and reduced 

tubercles. Although these effects in the fish lack specificity, they are supported 

by the mammalian responses. Tier 1 in vivo responses are not observed at the 

lower concentrations in organisms with an intact HPG-axis, presumably due to 

compensatory processes.

 Please comment on the how the agency has integrated different sources of 

data along a biological continuum to characterize this endocrine 

interaction and the transparency and reasonableness of the conclusion 

drawn.
Charge 2.6

 In each of the cases studies, there was a lack of anticipated 

complementary and redundant responses indicative of a chemical’s 

interaction with the thyroid signaling pathway. In the rat, there were T4 

changes that were either marginal or equivocal (Chemical A), or isolated 

organ weight changes (Chemicals J and S) or histopathological changes 

of the thyroid gland (Chemical J) that were not coincident with hormone 

changes. In the AMA, there were some isolated responses not 

necessarily indicative in terms of the endpoint specificity of a 

hypothalamic-pituitary-thyroid axis perturbation (Chemicals A and N). 

The agency considered the lack of complementarity and redundancy in 

responses to support a conclusion of no interaction with the HPT axis, 

and viewed these isolated responses insufficient to support a conclusion 

of an interaction with the thyroid signaling pathway.

 Please comment on the how the agency has characterized this 

endocrine interaction at different levels of biological organization, 

and the transparency and reasonableness of the conclusion drawn. 
Charge 2.7

 In the absence of Tier 1 data, OSRI was available for Chemical 

X that indicated effects on thyroid endpoints in the rat but the 

results were inconsistent within and among studies and there 

was no OSRI presented from amphibian studies. Because of 

studies that were not specifically validated to detect an 

interaction with the thyroid hormonal pathway, limited data, and 

ambiguous results, the potential for Chemical X to interact with 

the thyroid pathway cannot be excluded.

 Please comment on the how the agency has characterized 

this endocrine interaction at different levels of biological 

organization, and the transparency and reasonableness of 

the conclusion drawn.
Charge 3

 Based on all of the case study analyses, please provide overall comments on 

how the agency has employed its WoE guidance and characterized the evidence 

and conclusions and include in your response the following points:

a. How consistent and transparent the cases studies are in terms of 

documentation.

b. How adequately the agency has described the extent of complementarity

and redundancy of responses and has integrated and interpreted diverse 

lines of evidence across different biological levels of organization and 

taxa to reach preliminary conclusions regarding endocrine interactions.

c. How the agency has used OSRI data to further characterize the 

observations from EDSP Tier 1 assays in determining potential chemical 

interactions with the E, A, and T signaling pathways.

d. How the agency has considered the understanding of a chemical’s mode 

of action and how that informs the weight that is placed on Tier 1 

responses in the presence of uncertainties introduced by dose setting, 

overt toxicity, and portal of entry issues.