Use of Urinary β2-Microglobulin in the Assessment of the Health Risk from Environmental Cadmium Exposure
Round 1
Reviewer 1 Report
Comments and Suggestions for Authorsapplsci-3926569
This manuscript presents an investigation into the use of urinary β₂-microglobulin (β₂M) versus estimated glomerular filtration rate (eGFR) as endpoints for assessing cadmium (Cd)-induced nephrotoxicity. The manuscript is well structured and clearly and understandably written. Hence, the MS may be published following a minor revision, according to the following comments:
1. Introduction. The novelty of the work (and how it is filling the current gap) is missing. Has any similar study been published before? What difference does your work make? Also, there are a few explanations of the rationale for the study design.
2. Table 1: Add p-values for inter-group comparisons (e.g., age, hypertension). Include geometric means for skewed variables (Cd excretion).
3. The pictures in the manuscript are not pleasing to look at.
4. The Conclusions should include a statement highlighting the objective of the study that was met by this work. Moreover, this section should be written more inclusive and also conclude the significant contribution of the findings of the study for a particular application.
Author Response
Reviewer 1
Comments and Suggestions
applsci-3926569
This manuscript presents an investigation into the use of urinary β₂-microglobulin (β₂M) versus estimated glomerular filtration rate (eGFR) as endpoints for assessing cadmium (Cd)-induced nephrotoxicity. The manuscript is well structured and clearly and understandably written. Hence, the MS may be published following a minor revision, according to the following comments:
- We thank the reviewer for her/his comments, suggestions, and guidance to improve our paper. Accordingly, we have revised a manuscript to address issues raised. Changes to the text are in blue lettering.
Item 1. Introduction. The novelty of the work (and how it is filling the current gap) is missing. Has any similar study been published before? What difference does your work make? Also, there are a few explanations of the rationale for the study design.
Response: Abstract and Introduction have been rewritten to better reflect the main objective and novelty of the present study.
Item 2. Table 1: Add p-values for inter-group comparisons (e.g., age, hypertension). Include geometric means for skewed variables (Cd excretion).
Response: Statistical test for inter-group comparisons have been provided for age, and smoking prevalence, which showed significant variation across the eGFR groups. Geometric mean and geometric SD values have been provided for skewed variables that included excretion rates of Cd and β2M.
Item 3. The pictures in the manuscript are not pleasing to look at.
Response: Dose-response curves displayed in all figures are outputs of the PROAST software which are not completely modifiable. The main data have been extracted and presented in inserted tables.
Item 4. The Conclusions should include a statement highlighting the objective of the study that was met by this work.
Moreover, this section should be written more inclusive and also conclude the significant contribution of the findings of the study for a particular application.
Response: The conclusion has been revised to contain significant contributions with a special emphasis given to a new threshold identified, for the first time, from the eGFR endpoint using advanced BMD modeling. Please kindly note that results and discussion have been extensively revised. Results from additional data analysis to validate a proposed threshold are presented in supplementary material (Table S1).
Reviewer 2 Report
Comments and Suggestions for AuthorsI reviewed the manuscript entitled Use of Urinary β2-Microglobulin in the Assessment of the Health Risk from Environmental Cadmium Exposure.
I agree to accept this manuscript after major revision.
1) Keywords: β2-microglobulin as the first keyword, its first letter needs to be capitalized, so it should be changed to β 2-Microglobulin. Do not use abbreviations in keywords, simply use the full name, such as eGFR.
2) an excretion of β2-microglobulin (β2M) at a rate above 300 μg/ g creatinine, a tolerable Cd intake level was estimated to be 0.83 μg/kg body weight per day (58 μg/day for a 70 kg person), 300 μg/ g should change to 300 μg/g; 58 μg/day should change to 58 μg/d. Use international units instead of words. Check and modify similar issues throughout the entire text.
3) −80 °C should change to −80°C, there should be spaces between numbers and units, except for °C and %. Check and revise the entire text.
4) An alkaline (NaOH) solution was added to adjust the pH of urine aliquots, you can directly changed: An NaOH solution was added and indicate the concentration.
5) Figure 2. mg/ g should change to mg/g. 0.03 μg/ L should change to 0.03 μg/L. Check and modify similar issues throughout the entire text.
6) Figure 4. Eβ2M/Ccr level 2 was defined as (Eβ2M/Ccr) ×100 ≥ 300 μg/L filtrate. Ccr, All cr need to be changed to subscripts.
7) The text contains an excessive use of "we" and "our"; please revise to retain only a minimal number of these terms, enhancing the objectivity for readers.
8) The paper attributes the issues caused by creatinine correction to "reverse causality." Can a more intuitive analogy or schematic diagram be used in the introduction or discussion to explain how this statistical bias specifically occurs and why it leads to an underestimation of cadmium's true toxicity?
9) In the discussion, is it possible to directly compare the BMDL value you identified (0.17 µg/g creatinine) with the reference values for cadmium exposure proposed by other regions globally (such as the European Union's EFSA and the US ATSDR), thereby further highlighting the global public health significance of your research findings?
10) In your logistic regression model, you adjusted for age, BMI, sex, smoking, and hypertension. Were other potentially important confounding factors, such as prediabetes status, alcohol consumption, or history of non-steroidal anti-inflammatory drug use, considered and evaluated?
11) The BMDU/BMDL ratio is a valuable indicator of uncertainty. Could the comparison between the eGFR-based BMDL (low ratio, certain result) and the β₂-M-based BMDL (extremely high ratio, uncertain result) be presented more explicitly in the abstract or key results section, allowing readers to immediately grasp this core difference?
12) If the new threshold you proposed (0.20 µg/g creatinine) is adopted, what percentage of the current study cohort would be classified as at risk based on your data? Could this serve as a compelling argument for revising the current standards?
13) Your study is based on a Thai population. Given that the Thai diet is predominantly rice-based, the sources and patterns of cadmium exposure in this population may differ from those in Western populations, where exposure occurs mainly through wheat and vegetables. Could this affect the generalizability of your research findings? It is recommended to discuss this in the limitations section.
14) This study employs a cross-sectional design, which precludes the confirmation of causal relationships. In the discussion, could the association you identified be substantiated by citing literature from prospective cohort studies (such as the Swiss and Japanese studies you referenced), to support the likelihood of a causal link?
15) The most impactful finding of the paper—that the new threshold (~0.2 µg/g) is only about 1/25th of the old threshold (5.24 µg/g)—should be clearly emphasized and reiterated in the abstract, introduction, and discussion sections to ensure this core message is conveyed throughout the entire paper.
16) References, All references lack DOI numbers, please add them all.
17) The repetition rate of the paper is too high, and the author must revise and reduce it by at least 30%.
18) This study employed advanced benchmark dose (BMD) modeling in a Thai cohort (n=799) to reassess health risks from dietary cadmium exposure. The BMD limit for urinary cadmium was identified as 0.17 μg/g creatinine when using a reduction in estimated glomerular filtration rate (eGFR) as the endpoint, whereas no reliable BMD could be established using β₂-microglobulin excretion. Given that eGFR reduction is a more reliable indicator of chronic kidney disease, its use is recommended for deriving health-protective guidelines. The findings demonstrate that the current urinary cadmium threshold of 5.24 μg/g creatinine is inadequate, supporting the adoption of a threshold below 0.20 μg/g creatinine in future exposure guidelines.
Author Response
Reviewer 2
Comments and Suggestions
I reviewed the manuscript entitled Use of Urinary β2-Microglobulin in the Assessment of the Health Risk from Environmental Cadmium Exposure.
I agree to accept this manuscript after major revision.
- Thank you for your comments, suggestions, and guidance to improve our paper. Accordingly, we have revised an entire paper. Changes to the text are in blue lettering.
Item 1. Keywords: β2-microglobulin as the first keyword, its first letter needs to be capitalized, so it should be changed to β 2-Microglobulin. Do not use abbreviations in keywords, simply use the full name, such as eGFR.
Response: Suggested changes have been undertaken.
Item 2: an excretion of β2-microglobulin (β2M) at a rate above 300 μg/ g creatinine, a tolerable Cd intake level was estimated to be 0.83 μg/kg body weight per day (58 μg/day for a 70 kg person), 300 μg/ g should change to 300 μg/g; 58 μg/day should change to 58 μg/d. Use international units instead of words. Check and modify similar issues throughout the entire text.
Response: In its first appearance, an excretion of β2-microglobulin (β2M) at a rate above 300 μg/ g creatinine now reads an excretion of β2-microglobulin (β2M) at a rate above 300 μg/ g creatinine (cr). Later, it reads 300 μg/g cr. International abbreviations are adopted, where appliable
Item 3. −80 °C should change to −80°C, there should be spaces between numbers and units, except for °C and %. Check and revise the entire text.
Response: Required changes are undertaken.
Item 4. An alkaline (NaOH) solution was added to adjust the pH of urine aliquots, you can directly changed: An NaOH solution was added and indicate the concentration.
Response: The referred sentence now reads, “To prevent the degradation of β2M in acidic conditions, 1M NaOH was added to adjust the pH of urine aliquots to > 6.”
Item 5. Figure 2. mg/ g should change to mg/g. 0.03 μg/ L should change to 0.03 μg/L. Check and modify similar issues throughout the entire text.
Response: Abbreviation and spacing errors have been corrected, where applicable.
Item 6. Figure 4. Eβ2M/Ccr level 2 was defined as (Eβ2M/Ccr) ×100 ≥ 300 μg/L filtrate. Ccr, All cr need to be changed to subscripts.
Response: Required changes are undertaken
Item 7. The text contains an excessive use of "we" and "our"; please revise to retain only a minimal number of these terms, enhancing the objectivity for readers.
Response: The terms “we” and “our” are now used sparingly.
Item 8. The paper attributes the issues caused by creatinine correction to "reverse causality." Can a more intuitive analogy or schematic diagram be used in the introduction or discussion to explain how this statistical bias specifically occurs and why it leads to an underestimation of cadmium's true toxicity?
Response: The impact of total imprecision in the quantification of exposure and outcomes have been discussed fully in a paper by Grandjean and Budtz-Jørgensen (ref. 20). In the present study, the effects of Ecr correction for urine dilution are exemplified by data in Tables 2 and 3 plus the BMDU/BMDL ratios. We have rewritten subjection 4.1. Cd Exposure as a Risk Factor for CKD and Understated Toxicity by Ecr-Adjustment in the Discussion to better convey the message with one reference inserted (ref. 41) (lines 373-378)
.
[16] Grandjean, P.; Budtz-Jørgensen, E. Total imprecision of exposure biomarkers: Implications for calculating exposure limits. Am. J. Ind. Med. 2007, 50, 712-719.
[41] Horiguchi, H.; Oguma, E.; Sasaki, S.; Okubo, H.; Murakami, K.; Miyamoto, K.; Hosoi, Y.; Murata, K.; Kayama, F. Age-relevant renal effects of cadmium exposure through consumption of home-harvested rice in female Japanese farmers. Environ. Int. 2013, 56, 1-9.
Item 9. In the discussion, is it possible to directly compare the BMDL value you identified (0.17 µg/g creatinine) with the reference values for cadmium exposure proposed by other regions globally (such as the European Union's EFSA and the US ATSDR), thereby further highlighting the global public health significance of your research findings?
Response: In the original submission, reference to current dietary exposure guidelines was provided (ref. 6). In a revised version, a new subsection 4.4. Dietary Cd Exposure Carrying Negligible Health Risk, has been added to fully address the issues suggested by the reviewer (lines 419-441) with six additional references (ref. 46-51).
[6] Wong, C.; Roberts, S. M.; Saab, I. N. Review of regulatory reference values and background levels for heavy metals in the human diet. Regul. Toxicol. Pharmacol. 2022, 130, 105122.
[46] Satarug, S.; Swaddiwudhipong, W.; Ruangyuttikarn, W.; Nishijo, M.; Ruiz, P. Modeling cadmium exposures in low- and high-exposure areas in Thailand. Environ. Health Perspect. 2013, 121, 531-536.
[47] Satarug, S. Benchmark dose modeling to define permissible exposure levels for environmental cadmium. J. Environ. Expo. Assess. 2025, 4, 28.
[48]. Shi, Z.; Taylor, A. W.; Riley, M.; Byles, J.; Liu, J.; Noakes, M. Association between dietary patterns, cadmium intake and chronic kidney disease among adults. Clin. Nutr. 2018, 37, 276-84.
[49]. European Food Safety Authority (EFSA). Statement on tolerable weekly intake for cadmium. EFSA. J. 2011, 9, 1975.
[50] Schaefer, H.R.; Flannery, B.M.; Crosby, L.M.; Pouillot, R.; Farakos, S.M.S.; Van Doren, J.M.; Dennis, S.; Fitzpatrick, S.; Middleton, K. Reassessment of the cadmium toxicological reference value for use in human health assessments of foods. Regul. Toxicol. Pharmacol. 2023, 144, 105487.
[51] Swaddiwudhipong, W.; Limpatanachote, P.; Mahasakpan, P.; Krintratun, S.; Punta, B.; Funkhiew, T. Progress in cadmium-related health effects in persons with high environmental exposure in northwestern Thailand: a five-year follow-up. Environ. Res. 2012, 112, 194-198.
Item 10. In your logistic regression model, you adjusted for age, BMI, sex, smoking, and hypertension. Were other potentially important confounding factors, such as prediabetes status, alcohol consumption, or history of non-steroidal anti-inflammatory drug use, considered and evaluated?
Response: The inability to adjust for confounders the reviewer indicated has been declared in subsection 4.5. Strengths and Limitations (lines 442-465).
Item 11. The BMDU/BMDL ratio is a valuable indicator of uncertainty. Could the comparison between the eGFR-based BMDL (low ratio, certain result) and the β₂-M-based BMDL (extremely high ratio, uncertain result) be presented more explicitly in the abstract or key results section, allowing readers to immediately grasp this core difference?
Response: Required changes have been made to the text in the Discussion to address the reviewer’s suggestions.
Item 12. If the new threshold you proposed (0.20 µg/g creatinine) is adopted, what percentage of the current study cohort would be classified as at risk based on your data? Could this serve as a compelling argument for revising the current standards?
Response: Additional 4×4 tabulation and logistic regression have been conducted. Results are reported in subsection 3.6. Validity Analysis of a Proposed Urinary Cd Threshold (lines 354-360) and supplementary material (Table S1).
Item 13. Your study is based on a Thai population. Given that the Thai diet is predominantly rice-based, the sources and patterns of cadmium exposure in this population may differ from those in Western populations, where exposure occurs mainly through wheat and vegetables. Could this affect the generalizability of your research findings? It is recommended to discuss this in the limitations section.
Response: The generalizability issue has been discussed in subsection 4.5. Strengths and Limitations (lines 454-457).
Item 14. This study employs a cross-sectional design, which precludes the confirmation of causal relationships. In the discussion, could the association you identified be substantiated by citing literature from prospective cohort studies (such as the Swiss and Japanese studies you referenced), to support the likelihood of a causal link?
Response: Evidence for Cd/eGFR reduction causal link has now mentioned in subsection 4.5. Strengths and Limitations (lines 460-465). Reference to irreversibility of nephron destruction by Cd that causes eGFR to fall has been added (ref. 51).
[51] Swaddiwudhipong, W.; Limpatanachote, P.; Mahasakpan, P.; Krintratun, S.; Punta, B.; Funkhiew, T. Progress in cadmium-related health effects in persons with high environmental exposure in northwestern Thailand: a five-year follow-up. Environ. Res. 2012, 112, 194-198.
Item 15. The most impactful finding of the paper—that the new threshold (~0.2 µg/g) is only about 1/25th of the old threshold (5.24 µg/g)—should be clearly emphasized and reiterated in the abstract, introduction, and discussion sections to ensure this core message is conveyed throughout the entire paper.
Response: We appreciate the reviewer’s advice. We have made every effort to follow it through, where possible.
Item 16. References, All references lack DOI numbers, please add them all.
Response: It is understood that DOI numbers will be added by editorial staff.
Item 17. The repetition rate of the paper is too high, and the author must revise and reduce it by at least 30%.
Response: The similarity rate can be reduced to an acceptable level.
Item 18. This study employed advanced benchmark dose (BMD) modeling in a Thai cohort (n=799) to reassess health risks from dietary cadmium exposure. The BMD limit for urinary cadmium was identified as 0.17 μg/g creatinine when using a reduction in estimated glomerular filtration rate (eGFR) as the endpoint, whereas no reliable BMD could be established using β₂-microglobulin excretion. Given that eGFR reduction is a more reliable indicator of chronic kidney disease, its use is recommended for deriving health-protective guidelines. The findings demonstrate that the current urinary cadmium threshold of 5.24 μg/g creatinine is inadequate, supporting the adoption of a threshold below 0.20 μg/g creatinine in future exposure guidelines.
Response: Thank you for giving us the above paragraph. It has now replaced a second half of the abstract.
Reviewer 3 Report
Comments and Suggestions for AuthorsThis manuscript investigates the reliability of urinary β2-microglobulin (β2M) as an indicator of kidney damage due to environmental cadmium (Cd) exposure, employing benchmark dose (BMD) modeling and comparing β2M excretion with estimated glomerular filtration rate (eGFR) as toxicological endpoints. Using a well-characterized Thai cohort (n = 799), the authors propose that eGFR is a more accurate and clinically meaningful biomarker of early Cd nephrotoxicity than β2M, suggesting that current Cd exposure thresholds (e.g., 5.24 µg/g creatinine) significantly underestimate risk.
The manuscript essentially reanalyzes an existing cohort with alternative normalization and modeling approaches. While this is valuable, the novelty should be more clearly delineated. The current framing overstates originality. The claim that β2M is conceptually flawed as a biomarker should be supported by mechanistic or longitudinal evidence, not solely statistical modeling.
The Introduction should better integrate the findings within the context of established risk assessment frameworks. The discussion could benefit from referencing contemporary debates on low-level Cd nephrotoxicity thresholds.
Details such as the selection of dose–response models, convergence criteria, and BMR justification (5%) must be expanded in the Methods section.
The paper introduces Ccr-adjusted excretion as a corrective method. However, this adjustment is unusual for epidemiological biomonitoring. The authors must provide validation or cross-comparison with established normalization methods. The claim that Ccr adjustment “obliviates the need for timed urine collection” is overstated and requires empirical support.
How do the proposed thresholds compare with those derived from global biomonitoring data (e.g., NHANES, European HBM4EU)?
Is there evidence that eGFR reduction appears before tubular dysfunction markers such as β2M in longitudinal data?
Can the authors validate their creatinine clearance adjustment in an independent dataset or through comparison with 24-hour urine data?
How would applying the new 0.17 µg/g creatinine threshold affect population-level risk assessments globally?
Could the modeling approach be extended to include bone or cardiovascular endpoints, which are also linked to Cd exposure?
Percent match: 21% to this preprint https://doi.org/10.21203/rs.3.rs-6799604/v1
Author Response
Reviewer 3
Comments and Suggestions for Authors
This manuscript investigates the reliability of urinary β2-microglobulin (β2M) as an indicator of kidney damage due to environmental cadmium (Cd) exposure, employing benchmark dose (BMD) modeling and comparing β2M excretion with estimated glomerular filtration rate (eGFR) as toxicological endpoints. Using a well-characterized Thai cohort (n = 799), the authors propose that eGFR is a more accurate and clinically meaningful biomarker of early Cd nephrotoxicity than β2M, suggesting that current Cd exposure thresholds (e.g., 5.24 µg/g creatinine) significantly underestimate risk.
- We thank the reviewer for her/his comments, suggestions, and guidance to improve our paper. Accordingly, we have revised a manuscript to address issues raised. Changes to the text are in blue lettering.
Item 1. The manuscript essentially reanalyzes an existing cohort with alternative normalization and modeling approaches. While this is valuable, the novelty should be more clearly delineated. The current framing overstates originality. The claim that β2M is conceptually flawed as a biomarker should be supported by mechanistic or longitudinal evidence, not solely statistical modeling.
Response: Abstract and Introduction have been rewritten to better reflect objectivity and novelty of the present work. We specifically questioned the use of β2M excretion as a criterion to judge the nephrotoxicity of Cd and used it as a basis to derive safe dietary exposure levels.
Item 2. The Introduction should better integrate the findings within the context of established risk assessment frameworks. The discussion could benefit from referencing contemporary debates on low-level Cd nephrotoxicity thresholds.
Response: Please see response to item 1.
Item 3. Details such as the selection of dose–response models, convergence criteria, and BMR justification (5%) must be expanded in the Methods section.
Response: The various mathematical dose-response models we used have been scientifically scrutinized and they are recommended by the EFSA. All essential relevant references are provided.
Item 4. The paper introduces Ccr-adjusted excretion as a corrective method. However, this adjustment is unusual for epidemiological biomonitoring. The authors must provide validation or cross-comparison with established normalization methods. The claim that Ccr adjustment “obliviates the need for timed urine collection” is overstated and requires empirical support.
Response: Adjustment of urine concentration to creatinine clearance does not mean that one needs to do timed urine collection. It can be done by using an equation. We now provided the derivation of such equation in Supplemental Material. It requires a knowledge of plasma creatinine concentration, meaning blood sampling along with urine sampling. In the present study, results obtained from Ecr and Ccr were compared and the data speak themselves. Please See Tables 2 and 3.
Item 5. How do the proposed thresholds compare with those derived from global biomonitoring data (e.g., NHANES, European HBM4EU)?
Response: In a revised version, we have provided results of validity assessment of a proposed threshold by 4×4 tabulation and logistic regression in subsection 3.6. Validity Analysis of a Proposed Urinary Cd Threshold (lines 354-360) and supplementary material (Table S1).
Item 6. Is there evidence that eGFR reduction appears before tubular dysfunction markers such as β2M in longitudinal data?
Response: We have provided evidence for Cd/eGFR reduction causal link in subsection 4.5. Strengths and Limitations (lines 460-465). In addition, reference to irreversibility of nephron destruction by Cd that causes eGFR to fall has been added (ref. 51).
[51] Swaddiwudhipong, W.; Limpatanachote, P.; Mahasakpan, P.; Krintratun, S.; Punta, B.; Funkhiew, T. Progress in cadmium-related health effects in persons with high environmental exposure in northwestern Thailand: a five-year follow-up. Environ. Res. 2012, 112, 194-198.
Item 7. Can the authors validate their creatinine clearance adjustment in an independent dataset or through comparison with 24-hour urine data?
Response: We do not have data on 24-h creatinine excretion. We wish to encourage those have the data adopt this method of normalization. We have published a paper below that applied Ccr normalization to quantify an effect on Cd on eGFR.
Satarug, S.; Vesey, D.A.; Nishijo, M.; Ruangyuttikarn, W.; Gobe, G.C.; Phelps, K.R. The Effect of Cadmium on GFR Is Clarified by Normalization of Excretion Rates to Creatinine Clearance. Int. J. Mol. Sci. 2021, 22, 1762. https://doi.org/10.3390/ijms22041762
Item 8. How would applying the new 0.17 µg/g creatinine threshold affect population-level risk assessments globally?
Response: The most likely scenario is that CKD will continue to rise due to high prevalence of diabetes, obesity, and hypertension as it is projected [ref 14, 15]. Minimization of exposure from all sources, at very least, slow the rate of eGFR fall to kidney failure, requiring dialysis or kidney transplant.
Item 9. Could the modeling approach be extended to include bone or cardiovascular endpoints, which are also linked to Cd exposure?
Response: In theory, advanced BMD modeling can be applicable to any toxic endpoints. As the reviewer has pointed out, Cd has multiple toxicity endpoints, it is most important to define which end point is most sensitive to Cd? Cd exposure guideline derived from the most sensitive endpoint is protective against all other adverse effects.
Item 10. Percent match: 21% to this preprint https://doi.org/10.21203/rs.3.rs-6799604/v1
Response: The similarity rate can be reduced to an acceptable level.
Reviewer 4 Report
Comments and Suggestions for AuthorsDear authors,
The topic is important and is within the scope of the journal Applied Sciences.
Please provide the written informed consent that the participants had to sign.
Exclusion and inclusion criteria are not specified. Although you mention that this is a continuation from a previous work, this data is necessary.
There are many other factors that could affect your response like geographical site, co-exposures to other metals, and socioeconomic status that are not mentioned.
The abstract over-claims regarding regulatory implications, soften and add numerical uncertainity (BMDU/BMDL ratios) for the headline BMDL
Mention the CCR-based continuous-endpoint BMDL for eGFR in the text.
Check grammar details
-eHGFR instead of eGFR
-Neprhon instead of Nephron
-Model weighs or model weights?
- Krustal-Wallis's test or Krustal Wallis test
Author Response
Reviewer 4
Comments and Suggestions
Dear authors,
The topic is important and is within the scope of the journal Applied Sciences.
- Thank you for your comments and suggestions to improve our paper. Accordingly, we have extensively revised a manuscript. Changes to the text are in blue lettering.
Item 1. Please provide the written informed consent that the participants had to sign.
Response: The required item will be provided in compliance with the standard practice of journal (Appl. Sci).
Item 2. Exclusion and inclusion criteria are not specified. Although you mention that this is a continuation from a previous work, this data is necessary.
Response: An entire subjection 2.1 Data Sourcing has been rewritten. Exclusion and inclusion criteria have now been included along with other factors/considerations that may have influenced exposure and outcomes (lines 77-107). Six new references have been added (ref 23-26 and ref 28).
[23] Swaddiwudhipong, W.; Nguntra, P.; Kaewnate, Y.; Mahasakpan, P.; Limpatanachote, P.; Aunjai, T.; Jeekeeree, W.; Punta, B.; Funkhiew, T.; Phopueng, I. Human health effects from cadmium exposure: Comparison between persons living in cadmium-contaminated and non-contaminated areas in northwestern Thailand. Southeast Asian J. Trop. Med. Publ. Health 2015, 46, 133–142.
[24] Nishijo, M.; Suwazono, Y.; Ruangyuttikarn, W.; Nambunmee, K.; Swaddiwudhipong, W.; Nogawa, K.; Nakagawa, H. Risk assessment for Thai population: Benchmark dose of urinary and blood cadmium levels for renal effects by hybrid approach of inhabitants living in polluted and non-polluted areas in Thailand. BMC Publ. Health 2014, 14, 702.
[25] Suwatvitayakorn, P.; Ko, M.S.; Kim, K.W.; Chanpiwat, P. Human health risk assessment of cadmium exposure through rice consumption in cadmium-contaminated areas of the Mae Tao sub-district, Tak, Thailand. Environ. Geochem. Health 2020, 42, 2331–2344.
[26] Swaddiwudhipong, W.; Mahasakpan, P.; Limpatanachote, P.; Krintratun, S. Correlations of urinary cadmium with hypertension and diabetes in persons living in cadmium-contaminated villages in northwestern Thailand: A population study. Environ. Res. 2010, 110, 612–616.
[28] Zarcinas, B.A.; Pongsakul, P.; McLaughlin, M.J.; Cozens, G. Heavy metals in soils and crops in Southeast Asia. 2. Thailand. Environ. Geochem. Health 2004, 26, 359–371.
[29] Sripaoraya, K.; Siriwong, W.; Pavittranon, S.; Chapman, R.S. Environmental arsenic exposure and risk of diabetes type 2 in Ron Phibun subdistrict, Nakhon Si Thammarat Province, Thailand: Unmatched and matched case-control studies. Risk Manag Healthc. Policy 2017, 10, 41–48.
Item 3. There are many other factors that could affect your response like geographical site, co-exposures to other metals, and socioeconomic status that are not mentioned.
Response: Please see responses to item 2.
Item 4. The abstract over-claims regarding regulatory implications, soften and add numerical uncertainity (BMDU/BMDL ratios) for the headline BMDL
Response: Abstract has been rewritten to better reflect regulatory implications. BMDU/BMDL ratios are now mentioned in the Discussion and conclusion. Additional 4×4 tabulation and logistic regression have been conducted to argue for revising the current standards. Results are reported in subsection 3.6. Validity Analysis of a Proposed Urinary Cd Threshold (lines 354-360) and supplementary material (Table S1).
Item 5. Mention the CCR-based continuous-endpoint BMDL for eGFR in the text.
Response: Data on Ccr-based BMDL value (ECd/Ccr) for eGFR endpoint has now been discussed, where applicable.
Item 6.
Check grammar details
-eHGFR instead of eGFR
-Neprhon instead of Nephron
-Model weighs or model weights?
Krustal-Wallis's test or Krustal Wallis test
Response: Typo errors have been corrected.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe author has made the necessary modifications and explanations as per my request, therefore I agree to accept it in its current form.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors promplty responded to all of my inquiries.
