Overall, with the exception of the reviewer comments below, the AOP incorporates relevant  scientific literature and evidence, and the scientific content of the AOP reflects current scientific knowledge on this topic. The weight-of-evidence judgement/scoring (for each KER and the AOP as a whole) is well described and justified based on the evidence presented.

Reviewer 1:
Overall impression: well written, complete and plausible. Only minor revisions necessary.
Had some initial reservations about taxonomic applicability of differentiation of testis and ovary (KE1790, KER2146) since most of the data are fish, but in principle it's all vertebrates, as stated.

Reviewer 2:
...the AOP network is complete and plausible and the report is very well written.

Reviewer 3:
I don't have major concerns... just one on the ability of the undifferentiated gonad to synthesise
steroids. Everything else is well established. I found the literature cites a bit strange. Many key papers are missing whilst Angelopoulou’s one appears to be cited in most key paragraphs.

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Comments on "AOP Report--AOPs 346&376 Revised_Final.docx";
page, line number (reviewer number)

2.56 (R2)
"throughout the world to conduct risk assessments  and institute regulatory controls"
And in some regions regulatory controls based on hazard assessment (e.g. EU)

3.77 (R2)
"of the 350+ author- described AOPs in the AOP-Wiki"
Maybe include that the wiki is a part of the OECD supported Adverse Outcome Pathway Knowledge Base (AOP-KB) 
Also
"author described" to "author-described"

4.83 (R1)
"For example, 20 existing AOPs capture"
"For example, 20 AOPs in the AOP-Wiki capture"

6.143 (R3)
"In terms of KE 1789 (E2 synthesis by the undifferentiated gonad ), techniques exist "
All the techniques listed below are referring to a differentiated gonad.  I have never seen evidence for steroidogenesis in undifferentiated gonads. Please cite if available or rephrase if E2 is potentially produced in other tissues such as brain and/or gut (early life stages do have those tissues but do not have a functional gonad). 

10.256 (R2)
"in an experiment with fadrozole  using zebrafish genetic females "
Suggest to add that the genetic females were produced by mating of wild-type females with gynogenetic sex-reversed males because no marker of genetic sex in Zf is known.

11.279 (R1)
"In general , effects on downstream KEs occurred at concentrations equal to or greater than those at which upstream events occurred (Villeneuve et al. 2014 )."
If you say "In general", I immediately wonder what the exceptions are...
Is this reference correct? I can't find anything corresponding to this text in it.

11.285 (R1)
"That is, dependence of the severity of  the downstream in vivo responses on concentration and potency of chemicals activating the AR "
"severity" isn't a part of it, is it? I thought a key event either is or is not triggered...

11.291 (R1)
"synthetic steroids R1881 ›17α-methytestosterone›17α- and 17β-trenbolone"
to
"synthetic steroids R1881, 17α-methytestosterone and 17β-trenbolone"

12.306 (R3)
"(Piferrer and Donaldson 1993; Galvez et al. 1995; Morthorst et al., 2010; Baumann et al., 2014 )."
 Not sure why the FSDT validation report is not also cited here. Lots of tests involving regulatory species 

12.217 (R2)
"masculinization and feminization of fish (e.g., Piferrer et al. 1993 ). "
And Orn et al 2006 with feminization at the highest MT concentration

13.347 (R3)
"derived from fish in class Osteichthyes."
To my knowledge all fish, regardless the sex determination type, will respond to exogenous androgens by male biased ratios when exposure happens during critical developmental window. They also respond by means on intersex or complete sex reversal when adults. All I am trying to say is that AR activation is more straight forward, has a wider applicability domain and happens during any life stage, aromatase inhibition is more variable... 

13.347 (R2)
"derived from fish in class Osteichthyes."
to
"derived from fish in class Osteichthyes (bony fishes)."

13.360 (R1)
"Human pharmaceuticals (e.g., fadrozole, letrozole) that specifically inhibit CYP19 (Key Event 36) theoretically could occur in aquatic environments, although it is unlikely that contamination by these types of compounds would be widespread. Of more concern are... "
This is a sort of risk assessment sketch (e.g. "concern"), not a description of known stressors… Also further down in this paragraph, highlighted [lines 373-374]
R2: agree
R3: Ditto; all that needs to be done is start from Various conazole fungicides...

14.372 (R3)
"occurrence of the same fungicides from field monitoring studies and concluded that rarely would environmental concentrations of the conazoles be sufficient to cause masculinization "
Again, this is mostly discussing risk and should be reviewed

14.388 (R3)
"Finally , there are a wide variety of steroidal androgens used to purposely masculinize fish that could enter aquatic environments associated with aquacultural operations (Pandian and Sheela 1995)."
I would add here synthetic progestins such as levonorgestrel, as they are by far the most potent androgenic xenobiotics. 

14.392 (R1)
"There is at least one nonchemical stressor that can influence AOP 346, temperature," 
to
"...AOP 346, namely temperature," 

14.394 (R2)
"has been clearly demonstrated in various fish (and  amphibian) species"
reptile!

15.404 (R2)
"The AOP framework directly supports the risk  assessment/"
And hazard assessment

16.430 (R2)
"describe a 42-d fish sexual development   test"
This was the draft version of the FSDT (OECD TG 234) so you could refer to that one, including also Japanese medaka and three-spined stickleback
R3: I made a similar comment further up

16.446 (R2)
"This, in turn, provides the basis for species and endpoint selection for additional testing and monitoring "
Also, the AOPs can support development/refinement of endpoints in test guidelines (OECD, USEDSP etc) to better predict adversity/MoA 

25.689 (R1)
"Society for the Advancement of Adverse Outcome Pathways (SAAOP). 2022. Welcome to the Collaborative Adverse Outcome Pathway Wiki (AOP-Wiki). Available from: https://aopwiki.org/.  Last accessed 2 August 2022."
to
"SAAOP. 2022. Society for the Advancement of Adverse Outcome Pathways. Welcome to the Collaborative Adverse Outcome Pathway Wiki (AOP-Wiki). Available from: https://aopwiki.org/ and http://www.saaop.org/. Accessed 2 August 2022."
(to correspond to text citations to SAAOP 2022)

26.704 (R1)
"(EDSP) overview. Washington, DC. https://www.epa.gov/endocrine-disruption/endocrine-disruptor-screening-program-edsp-overview"
to
"...endocrine-disruptor-screening-program-edsp-overview. Accessed 2 August 2022"

26.725 (R1)
"WHO/PCS/EDC/02.2."
to
"WHO/PCS/EDC/02.2. https://www.who.int/publications/i/item/WHO-PSC-EDC-02.2 . Accessed 14 November 2022"


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Comments on 
"AOP 346 Snapshot-2022-09-12T11_50_04+00_00.pdf":
(p=page number, R=reviewer number)

p.1 (R2)
Short Title: Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation
Should "fish" or "teleost fish" be a part of the title? 

p.1 (R2)
Background
"In fish sexual differentiation occurs post hatch and can be influenced by exogenous factors such as chemicals, temperature, pH, population density, social cues and more. As a result, the gonadal sex phenotype in many fish can be altered by environmental conditions experienced during development..."
to "in oviparous fish"

p.2 (R2)
"Inhibition of CYP19a1 expression or activity"
to "...CYP19a..."

p.2 (R1)
"when estrogen signaling is reduced there is a a resultant bias"
to 
"when estrogen signaling is reduced there is a resultant bias"

p.4 (R1)
"indicated that E2 treatment could could restore a near normal sex ratio defect"
to
"indicated that E2 treatment could restore a near normal sex ratio"

p.4 (R1)
"and ultimatelywere fertile"
to
"and ultimately were fertile"

p.4 (R3)
 [Table] E2 Synthesis by the undifferentiated gonad "moderate"
Is this weak or moderate? See text under uncertainties, incosistencies, page 6/38

p.5 (R1)
Concordance of Dose Response Relationships
"Most of these studies only measured one key event in the AOP so cannot be directly used to explore dose-response concordance between key event relationships"
to
"...concordance between key events"?

p.5 (R1)
Overall Assessment of the AOP; Concordance of Dose Response Relationships
"The differential sensitivity to inhibition of aromatase .... rank order potency of the test chemicals was
generally similar."
This paragraph is not about dose response relationships. Suggest move to KE36 Inhibition, Aromatase (=MIE);
Domain of Applicability; Taxonomic Applicability.

p.14 (R1)
How it is Measured or Detected
"Spitzbergen" to "Spitsbergen"

p.15 (R2)
Event: 1791: Increased, Male Biased Sex Ratio
Key Event Description
"at some point a male-biased population could become problematic"
to
"at some point a female-biased population could become problematic"

p.15 (R2)
Event: 1791: Increased, Male Biased Sex Ratio
Key Event Description
"exposure during gonadal differentiation to androgen receptor antagonists or inhibitors of cytochrome P450 19a1"
to
"...androgen receptor agonists or..."

p.16 (R1)
"List of Adverse Outcomes in this AOP"
I thought an AOP has by definition got only one MIE, so shouldn't it just be "MIE" instead of "List of MIEs"?

p.33 (R2)
Relationship: 2167: Inhibition, Aromatase leads to Increased, Differentiation to Testis
Evidence Supporting this KER
"Frenske et al. 2004"
to
"Fenske..."

p.34 (R1)
Relationship: 2167: Inhibition, Aromatase leads to Increased, Differentiation to Testis
Uncertainties and Inconsistencies
"As noted below, it is uncertain as to the full range of species this key event relationship might be applicable
due to susbtantial taxonomic variation in the role that steroid signaling plays in gonadal differentiation."
is difficult to read (also "as note below" is not clear"); suggest change to
"Due to substantial taxonomic variation in the role that steroid signaling plays in gonadal differentiation, the
range of species that this key event relationship applies to is uncertain."

p.37 (R2)
Relationship: 2350: Inhibition, Aromatase leads to Increased, Male Biased Sex Ratio
Empirical Evidence
"Frenske et al. 2004"
to
"Fenske..."

p.37 (R2)
Relationship: 2350: Inhibition, Aromatase leads to Increased, Male Biased Sex Ratio
Empirical Evidence
"Exposure of fathead minnows (Pimephales promelas) to the aromatase inhibitor prochoraz"
to
"Exposure of fathead minnows (Pimephales promelas) and zebrafish (Danio rerio) to the aromatase inhibitor prochoraz"

p.37 (R1)
Relationship: 2350: Inhibition, Aromatase leads to Increased, Male Biased Sex Ratio
Uncertainties and Inconsistencies
"As noted below, it is uncertain as to the full range of species this key event relationship might be applicable due to susbtantial taxonomic variation in the role that steroid signaling plays in gonadal differentiation."
is difficult to read (also "as note below" is not clear"); suggest change to
"Due to substantial taxonomic variation in the role that steroid signaling plays in gonadal differentiation, the range of species that this key event relationship applies to is uncertain."

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Comments on 
"AOP 376 Snapshot-2022-09-12T11_51_24+00_00.pdf":

p.1 (R2)
Short Title: AR agonism leading to male-biased sex ratio
Should "fish" or "teleost fish" be included in the title?

p.2 (R2)
Taxonomic Applicability
"zebra-fish" to "zebra fish"

p.3 (R1)
Overall Assessment of the AOP
"induced a male biased sex ratio in a dose-dependent manner.."
to
"induced a male-biased sex ratio in a dose-dependent manner."
(Double full stop "..")

p.3 (R1)
Overall Assessment of the AOP
"could result ina male biased sex ratio"
to
"could result in a male-biased sex ratio"

p.3 (R2)
Essentiality of the Key Events
"could result ina male biased sex ratio"
to
"could result in a male biased sex ratio"

p.4 (R2)
Essentiality of the Key Events
"exposure to AR agonsts"
to
"...agonists"

p.4  (R2)
Essentiality of the Key Events
"Miller et al. 2021" should be "Miller et al. 2022" according to References

p.4 (R1)
Overall Assessment of the AOP
Title "Concordance of Dose Response" to "Concordance of Dose Response Relationships"

p.4  (R2)
Concordance of Dose Response
"Miller et al. 2021" should be "Miller et al. 2022" according to References

p.4 (R2)
Concordance of Dose Response
"increased number of males in a dose-dependent fashion (Orn et al., 2003; Morthorst et al., 2010; Baumann et al., 2013; Baumann et al., 2014)"
Also Holbech et al., 2006 (all-male population from 9.7 ng/l trenbolone)

p.4 (R2)
Temporal concordance
"cannot be observed untl the"
to
"...until..."

p.5 (R2)
Consistency
"There are cases however, in which exposure to aromatizable androgens such as methyltestosterone can lead both to masculinization and feminization of fish (e.g., Piferrer et al. 1993); this..."
Also Orn et al 2003 where highest concentration of MT caused "feminization"

p.7 (R1)
Appendix 1; "List of MIEs in this AOP"
I thought an AOP has by definition got only one MIE, so shouldn't it just be "MIE" instead of "List of MIEs"?

p.12 (R1)
Event: 1790: Increased, Differentiation to Testis
How it is Measured or Detected
"(e.g., Spitzbergen et al. 2009)."
to
"(e.g., Spitsbergen et al. 2009)."

p.12 (R2)
Event: 1791: "Increased, Male Biased Sex Ratio"
Title: "Increased" and "male biased" seem as double confect! Wouldn't "Male biased Sex Ratio" be clear?

p.13 (R2)
Event: 1791: Increased, Male Biased Sex Ratio
Domain of Applicability
"Any sexually reproducing organism can theoretically experience a male-biased population"
Decide on "male-biased" or "male biased"

p.13 (R2)
Event: 1791: Increased, Male Biased Sex Ratio
How it is Measured or Detected
"Fundamentally, determination of sex ratio (and consequently male-biased sex ratio) is based on counts of the number of males and/or females in a population, or some statistically representative sub-sample of a population."
Agree that sex ratio is based on counts of males and females but there are cases where the ratio is more relevant to calculate as male vs non-male and female vs non-female! for example where delayed development/arrested development causes a high number of undifferentiated fish. This is most relevant during chemical testing in e.g. TG 234, 240 FFLCT. Not sure if this should be noted here!

p.14 (R2)
Event: 1791: Increased, Male Biased Sex Ratio
How it is Measured or Detected
"It is not uncommon for published papers to report skewed sex ratios based on sample sizes far too small to result in meaningful conclusions."
Environmentally meaningful or? If a study show significant skewed sex ratio due to chemical exposure, guess this endpoint could be assessed as other significant endpoints!

p.20 (R1)
Relationship: 2260: Agonism, Androgen receptor leads to Increased, Differentiation to Testis
Uncertainties and Inconsistencies
"As noted below, it is uncertain as to the full range of species this key event relationship might be applicable due to susbtantial taxonomic variation in the role that steroid signaling plays in gonadal differentiation."
to
"Due to substantial taxonomic variation in the role that steroid signaling plays in gonadal differentiation, the range of species that this key event relationship applies to is uncertain."

p.20 (R2)
Relationship: 2260: Agonism, Androgen receptor leads to Increased, Differentiation to Testis
Empirical Evidence
"to determine gonad maturation and sperm stage (Orn et al., 2006; Morthorst et al., 2010 Baumann et al.,
2015).
also Holbech et al., 2006

p.20 (R1)
Relationship: 2260: Agonism, Androgen receptor leads to Increased, Differentiation to Testis
Uncertainties and Inconsistencies
"As noted below, it is uncertain as to the full range of species this key event relationship might be applicable due to susbtantial taxonomic variation in the role that steroid signaling plays in gonadal differentiation."
The paragraph is difficult to read (also "As noted below" is a problem); suggest change to
"Due to substantial taxonomic variation in the role that steroid signaling plays in gonadal differentiation, the range of species that this key event relationship applies to is uncertain."

p.22 (R2)
Relationship: 2146: Increased, Differentiation to Testis leads to Increased, Male Biased Sex Ratio
Empirical Evidence
"Frenske et al. 2004" to "Fenske et al. 2004"  

p.25 (R2)
Relationship: 2147: Increased, Male Biased Sex Ratio leads to Decrease, Population growth rate
Empirical Evidence
"vivipara)can" to "vivipara) can"

p.27 (R2)
Relationship: 2349: Agonism, Androgen receptor leads to Increased, Male Biased Sex Ratio
Empirical Evidence
"Controlled expoure of zebrafish (Danio rerio) to the synthetic androgen 17β-trenbolone during development has been shown to result in male biased sex ratios (Orn et al., 2006; Larsen & Baatrup, 2010; Morthorst et al., 2010; Baumann et al., 2013, 2015; Golan & Levavi-Sivian 2014)."
also Holbech et al., 2006

p.27 (R2)
Relationship: 2349: Agonism, Androgen receptor leads to Increased, Male Biased Sex Ratio
Empirical Evidence
"17β-trenbolone with with 17alpha-ethynyl estradiol admistered via"
to
"17β-trenbolone with 17alpha-ethinylestradiol..."

p.27 (R2)
Relationship: 2349: Agonism, Androgen receptor leads to Increased, Male Biased Sex Ratio
Empirical Evidence
"Exposure to methyltestosterone reulted in male biased cohorts"
to
"...resulted..."

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