Dear Academic Editor and Reviewers,

The authors thank you for taking the time to go through the manuscript and for providing comments that have improved the quality of the manuscript. Each comment and question have been addressed below.

Sincerely,

Dr. Silje Modahl Johanson

Reviewers' comments:

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: No

The authors thank the reviewers for taking the time to go through the manuscript. We agree that the conclusions should be moderated, particularly on the role on carcinogenesis. The manuscript has been edited accordingly.

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

The statistical analyses have been performed to the best of our abilities and we hope the reviewers are satisfied with the description presented in the manuscript.

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: No

For additional transparency, all raw data have been deposited in the online repository DataverseNO and can be found at https://dataverse.no/privateurl.xhtml?token=e2d40b17-f2dc-4974-8e6e-d0a44d5ffbe7. This is a private URL that can be used during the reviewing process and the dataset will be given the DOI https://doi.org/10.18710/MSTY0R when and if the manuscript is accepted for publishing. The deposited dataset is identical to ‘S9 Raw data’ originally submitted for review.

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

The authors have had the manuscript checked by a native English speaker and we hope the reviewers now find it presented in an intelligible fashion and written in standard English.

5. Review Comments to the Author

Reviewer #1:

In this paper, the authors have investigated the effects of a perinatal exposure to a mixture of POPs on mammary gland and ovarian folliculogenesis in mice. The design of the study is good, in accordance with OECD guidelines. The authors chose only 2 doses: low vs high. It could be considered as restrictive but the design of a F2 study is clearly too complex to consider more doses to investigate. They well discussed the limits and the bias of their work, especially regarding the ovarian function (no estrous cycle homogeneity, no quantification of follicle atresia, no evaluation of hormone levels).

The authors thank the reviewer for taking the time to go through the manuscript and for the positive feedback.

Some interrogations remain to consider to my opinion, and could enhance the discussion of the paper:

- how the authors can explain the lack of effect of high dose on BW of animals?

It is not clear why we did not observe any effect of the high dose on body weight. However, as the CD-1 mouse strain is an outbred laboratory strain it generally has a higher baseline variation in response to stimuli than inbred mouse strains. This could have caused the possible negative effects on body weight to be masked by natural variation and, thus, not showed significant differences here. Furthermore, the individual concentrations of POPs in the High dose were predominantly lower than the no observed adverse effect levels (NOAEL), where such levels were available [1]. Thus, despite this being a High dose compared to human consumption, the relatively more rapid xenobiotic metabolism of mice compared to humans could possibly explain the lack of toxic effect observed. A short discussion has been added to the manuscript on page 30, lines 611-615 in the Revised Manuscript with Track changes.

- how they can explain the no detection of HBCD in dams liver?

The LOD of HBCD was relatively high (1.182 ng/g wet weight) compared to the detection limit of the other POPs. Thus, it is possible that some of the samples had concentrations of HBCD just below the LOD. Furthermore, as the chemical analysis was conducted only on one pooled liver sample from each generation, gender and exposure group, it is possible that some of the individual samples had higher HBCD concentrations than others, and, when pooling the samples, the overall concentration was diluted by samples with lower HBCD concentrations.

The commercial mixture of HBCD consists of the three main diastereomers α, β and γ-HBCD, with the γ-HBCD being the predominating diastereomer comprising ≥ 70% [2]. In adult female mice, γ-HBCD has been shown to be rapidly metabolized and eliminated with a terminal half-life of approximately 4 days [3]. Thus, particularly for the offspring, it is possible that most HBCD had been eliminated from the body prior to sampling. However, this does not explain why dam livers continuously exposed to the mixture of POPs did not show higher levels of HBCD. Thus, the reason for the lack of HBCD in dam livers is unknown.

To my opinion, the authors must moderate their conclusion on the role on carcinogenesis, as their results did not well illustrate this point.

The authors agree with the reviewer and have moderated the conclusions on the role of the mixture of POPs on carcinogenesis. This can be seen on page 5 (line 55), page 28-29 (lines 571-575) and page 31 (line 624) in the Revised Manuscript with Track Changes.

In the introduction of the paper, the authors cited a lot of papers (55!). They should reduce this number and add some publications regarding humans.

The number of cited papers in the introduction has been reduced to 39 and more emphasis has been put on the Scientific Statements of The Endocrine Society [4, 5], which extensively reviews the existing literature on how exposure to EDCs may lay the foundation for various diseases later in life in both animals and humans.

Reviewer #2:

This study evaluates the impact of POP mixtures on mammary gland development, ovarian physiology and liver toxicity. However, it lacks novelty. Lack of hormonal analyses to support the results. The conclusions are based on speculations or hypotheses.

The authors thank the reviewer for taking the time to go through the manuscript.

In the study we utilized a mixture of POPs that is highly relevant to humans due to its complexity and inclusion of compounds from multiple chemical classes. Furthermore, we applied a human relevant route of exposure and discovered effects on multiple organs that could lead to adverse effects later in life. Other research articles using the same mixture of POPs have been well received in the scientific community [6-9]. To the authors knowledge, no other study has previously presented simultaneous results on female reproductive organs and liver caused by exposure to a large mixture of POPs.

The authors strongly agree that hormonal analyses should have been conducted to support the results. However, due to the design of the experiment, only single hormone measurements would have been possible. This would not have given a thorough characterization of the impact of the mixture of POPs on hormone levels, or the impact of hormones on mammary gland development. A better option would have been to chemically synchronize estrous cycle of all female offspring. However, this could possibly have compromised the aim of the experiment and concealed the effects of the mixture of POPs. Another option would have been to use vaginal smear assessment to euthanize mice at identical timepoints in their estrous cycle. However, this would have been very time consuming and not possible given the large number of mice included in the experiment. As commented by Reviewer 1, we have discussed these limitations in the manuscript (page 28, lines 555-556 and 564-566). For additional emphasis on the need for hormonal analyses, the last sentence of the abstract (page 2-3, lines 54-55) and the last paragraph of the discussion (page 31, lines 621-623) has been modified.

Other studies using the same mixture of POPs have shown effects on stress response, learning, memory, and altered gene expression patterns of hippocampal genes involved in cognitive function and neuroinflammation in female hybrid 129:C57BL/6 mice and their maternally exposed offspring [8, 10]. Additionally, testicular development, sperm production and sperm chromatin integrity were affected in maternally exposed male hybrid 129:C57BL/6 mice [9]. The mixture of POPs has also been shown to affect intestinal tumorigenesis in a susceptible animal model [6, 7], and alter blood lipid profiles in non-obese diabetic mice [11].

In addition to the mixture of POPs used in the present manuscript, Berntsen and colleagues [1] also created a mixture of POPs, as well as several sub-mixtures, for use in in vitro studies. This mixture was based on chemical concentrations in human blood found in the general Scandinavia population. This in vitro POP mixture has shown effects on nerve and immune cells [12-15], as well as inducing hyperactivity in zebrafish larvae [16, 17]. The total POP mixture or sub-mixtures have also been shown to act antagonistically on the aryl hydrocarbon receptor (AhR) in three different transgenic cell lines [18], and to antagonize transactivation and translocation of the androgen receptor in transfected androgen receptor cell lines [19], to affect in vitro secretion of glucagon-like peptide 1 (GLP-1) in an GLP-1 secreting enteroendocrine cell line [20] and to disrupt steroidogenesis at higher concentrations in H295R cells [21].

In the present manuscript, the conclusions have been moderated throughout the discussion. As commented by Reviewer 1, the design of the study is good and in accordance with OECD guidelines. It included a relatively high number of mice within each exposure group and sampling time. Furthermore, the conclusions have been drawn based on results from conservative statistical methods and, thus, the authors believe that the results presented are not based on speculations. However, as pointed out by both Reviewers, it is important to properly discuss the limitations of the study and we hope that the Reviewer is satisfied with the modifications implemented.

References

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9. Khezri A, Lindeman B, Krogenæs AK, Berntsen HF, Zimmer KE, Ropstad E. Maternal exposure to a mixture of persistent organic pollutants (POPs) affects testis histology, epididymal sperm count and induces sperm DNA fragmentation in mice. Toxicol Appl Pharmacol. 2017;329:301-8. doi: 10.1016/j.taap.2017.06.019.

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