The Navigation Guide—Evidence-Based Medicine Meets Environmental Health: Systematic Review of Nonhuman Evidence for PFOA Effects on Fetal Growth

Background: In contrast to current methods of expert-based narrative review, the Navigation Guide is a systematic and transparent method for synthesizing environmental health research from multiple evidence streams. The Navigation Guide was developed to effectively and efficiently translate the available scientific evidence into timely prevention-oriented action. Objectives: We applied the Navigation Guide systematic review method to answer the question “Does fetal developmental exposure to perfluorooctanoic acid (PFOA) or its salts affect fetal growth in animals ?” and to rate the strength of the experimental animal evidence. Methods: We conducted a comprehensive search of the literature, applied prespecified criteria to the search results to identify relevant studies, extracted data from studies, obtained additional information from study authors, conducted meta-analyses, and rated the overall quality and strength of the evidence. Results: Twenty-one studies met the inclusion criteria. From the meta-analysis of eight mouse gavage data sets, we estimated that exposure of pregnant mice to increasing concentrations of PFOA was associated with a change in mean pup birth weight of –0.023 g (95% CI: –0.029, –0.016) per 1-unit increase in dose (milligrams per kilogram body weight per day). The evidence, consisting of 15 mammalian and 6 nonmammalian studies, was rated as “moderate” and “low” quality, respectively. Conclusion: Based on this first application of the Navigation Guide methodology, we found sufficient evidence that fetal developmental exposure to PFOA reduces fetal growth in animals. Citation: Koustas E, Lam J, Sutton P, Johnson PI, Atchley DS, Sen S, Robinson KA, Axelrad DA, Woodruff TJ. 2014. The Navigation Guide—evidence-based medicine meets environmental health: systematic review of nonhuman evidence for PFOA effects on fetal growth. Environ Health Perspect 122:1015–1027; http://dx.doi.org/10.1289/ehp.1307177


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Authors did not respond to requests for additional information. Failure to report pertinent study details resulted in exclusion from quantitative analysis for birth weight outcome, despite meeting inclusion criteria otherwise. Authors did not specify chemical form of PFOA (assume CAS# 335-67-1). For prenatal time point, no significant effects on survival. For postnatal time point, delayed delivery, high rate of stillborn birth (58%), and remaining live pups died within 6 hours after birth for 10 mg/kg PFOA group; 16% of neonates died in 5 mg/kg PFOA group.  • 5, 5, 5, 4 for control, 0.1, 1, 5 mg/kg PFOA groups, respectively 2. Birth weight (g) -one individual pup from each litter weighed at PND1 (day of birth or birth occurred previous night). Number of dams analyzed: • 5, 4, 4, 5 for control, 0.1, 1, 5 mg/kg PFOA groups, respectively Not included in review: PFOA serum concentration prior to birth; dam PFOA serum concentrations; pup PFOA serum concentrations Notes Author responded to requests for additional information. Raw data provided by study author. Number of live fetuses and litter sizes at birth were statistically equal across doses and control groups.  White et al. 2009 (study ID 312).

Study Element Description Methods
Mouse developmental toxicological study Participants Wild-type CD-1 mice Timed-pregnant GD0 animals obtained from supplier Total number of dams allocated: 112 Exposure Dams treated with PFOA, ammonium salt (CAS# 3825-26-1) via daily gavage from GD8 to GD17. At birth, pups were cross-fostered to obtain the following groups: 1) never exposed, 2) exposed in utero and via lactation, 3) exposed only in utero, 4) exposed only via lactation. Exposure groups: • 1 dose groups = 5 mg PFOA/kg body weight/day; 56 dams • 1 control group = deionized water; 56 dams Outcomes 1. Birth weight (g) -3 individual female pups from each litter weighed at PND1 (at least 12 hours after birth and cross-foster); only 2 groups of pups relevant for review: 1) never exposed, 2) exposed in utero and via lactation. Number of dams analyzed: • 4 for each exposure group Not included in review: 2 additional studies -late-life effects cross-foster study and restrictedexposure study; mammary gland development scores; circulating serum PFOA concentration; mammary gland differentiation in offspring; serum PFOA dosimetry Notes Author responded to requests for additional information. Raw data provided by study author. Litter sizes were statistically equal across doses and control groups. • 19, 10, 7, 9, 13 for 0.1, 0.3, 0.6, 1 mg/kg PFOA groups, respectively Not included in review: studies of PPARα knockout mouse model; maternal weight and reproductive outcomes; liver weight in dams and offspring; offspring survival, development, and growth; serum PFOA in dams and offspring Notes Author responded to requests for additional information. Raw data provided by study author. Study authors noted that the 129S1/SvlmJ strain is more sensitive to PFOA exposure than other strains, such as CD-1 strain mice. Litter sizes were statistically equal across dose groups up to 1 mg/kg PFOA and control groups; incidence of full litter resorption was statistically significantly higher in 5 mg/kg PFOA groups (83, 80, 100% of dams had full litter resorption for 5, 10, 20 mg/kg PFOA groups, respectively).  White et al. 2007 (study ID 566).

Notes
Author responded to requests for additional information. Raw data provided by study author. Litter sizes and number of uterine implantation sites were statistically equal across doses and control groups. Author responded to requests for additional information. Raw data provided by study author. Litter sizes were statistically equal across doses and control groups. Incidence of whole litter loss was statistically significantly increased in the 5 mg/kg PFOA group as compared to other treatment groups (not caused by litter resorptions as statistically equal number of uterine implantation sites as compared to control).   • Inhalation: 23, 24, 23, 15, 7, 6, 5 for 0, 0.1, 1, 10, 25 mg PFOA/m 3 , pair-fed to 10 mg/m 3 , pair-fed to 25 mg/m 3 groups, respectively. • Gavage: 24, 22 for 0, 100 mg/kg PFOA groups, respectively. 2. Birth weight (g) -individual pups weighed at PND1 (day of birth). Number of dams analyzed: • Inhalation: 18, 10, 11, 6, 9 for 0, 0.1, 1, 10, 25 mg PFOA/m 3 groups, respectively.
• Gavage: 12, 9 for 0, 100 mg/kg PFOA groups, respectively. Not included in review: fetal teratology; dam body and liver weights; offspring survival; dam survival; offspring defects. Notes Study authors contacted to provide additional information, but information could not be obtained due to length of time since study conducted. Incidence of litter resorptions and litter size at birth were statistically equal across doses and control groups. Dams treated with PFOA (CAS# 333-67-1) via daily gavage from GD7-GD20/21. Exposure groups: • 1 dose groups = 20 mg PFOA/kg body weight/day; 8 dams • 1 control group = corn oil; 10 dams Outcomes 1. Fetal weight (g) -fetuses weighed individually at GD20/21. Number of dams analyzed: • 5, 6 for 0, 20 mg/kg PFOA groups, respectively. Not included in review: outcomes for treatment with diisobutyl phthalate and butylparaben; steroid hormone measurement; plasma levels of metabolic chemicals; mRNA expression; P450c17 and PPARγ protein levels in testes.

Notes
Author responded to requests for additional information. Raw data provided by study author; published study does not present data on this outcome. Author noted that "some animals were sacrificed one day too early for their age. Therefore, in the GD21 group, about one fourth are GD20 and three fourths are GD21". Study did not discuss fetal mortality. Dams treated with PFOA applied to food from GD1-GD20. Exposure groups: • 1 dose groups = 0.3 mg PFOA/kg body weight/day; 10 dams • 1 control group = food applied with ethanol; 6 dams Outcomes 1. Birth weight (g) -pups weighed at PND 1 (not clearly defined). Number of dams analyzed: • 6, 9 for 0, 0.3 mg/kg PFOA groups, respectively. Not included in review: outcomes from treatment with PFOS; PFOA concentrations in tissues; locomotor and exploratory activity; circadian activity; anxiety-related behavior; depression-like behavior; muscle strength; motor coordination Notes Author responded to requests for additional information. Raw data provided by study author; published study does not present data on this outcome. The author reported results for a larger number of PFOA-treated dams than originally allocated in the paper. Results from the study author do not clearly explain if the body weights are litter averages. Failure to report pertinent study details resulted in exclusion from quantitative analysis for birth weight outcome, despite meeting inclusion criteria otherwise. Authors did not specify chemical form of PFOA (assume CAS# 335-67-1). Litter sizes were statistically equal across doses and control groups. Author responded to requests for additional information. Raw data provided by study author; published study does not present data on this outcome. Published study included groups treated with drinking water containing PFOA, but study authors did not provide data for these studies and stated that only the groups treated via gavage could be compared. The number of uterine implantation sites was statistically equal across doses and control groups. Litter size at birth and prenatal survival were statistically significantly decreased in 5 mg/kg PFOA groups. Author responded to requests for additional information. This study was an industry report performed according to good laboratory practices (GLP). The report supports papers in peerreviewed literature. Litter sizes at birth were statistically equal across doses and control groups.  • 38, 35, 25 for control, 100, 500 µM PFOA in food groups, respectively Not included in review: lifespan, behavior, numbers of emerging progeny, lethality, developmental progress, effects of nutrient supplementation Notes Author responded to requests for additional information. Raw data provided by study author (including length measurements plotted for analysis). Emergence of progeny was statistically significantly reduced in both PFOA dose groups (24% and 73% decrease compared to control group for 100, 500 µM PFOA in food groups, respectively). Eggs injected with PFOA (CAS# 335-67-1) at incubation day 0. Exposure groups: • 2 dose groups = 5, 10 mg PFOA/kg egg • 1 control group = saline Outcomes 1. Hatchling weight (g) -individual hatchlings weighed 24 hours after hatching. Number of hatchlings analyzed: • 30, 12, 10 for control, 5, 10 mg PFOA/kg egg groups, respectively Not included in review: outcomes from treatment with PFOS; hatching and survival; morphological and functional scores; imprinting scores; protein kinase C concentrations in the intermedial part of the hyperstriatum ventrale Notes Author responded to requests for additional information. Study author provided data estimates used to create figure in paper. On incubation day 19, survival of embryos was statistically significantly reduced (approx 45-55%) in the PFOA treated groups as compared to controls; hatching was statistically significantly reduced (70-80%) compared to controls.  • 26, 10, 11, 12, 9 for uninjected, sunflower oil only, 0.5, 1, 2 mg PFOA/kg egg groups, respectively 2. Crown to rump length (mm) Hatchling time point: individual hatchlings measured 16-24 hours after hatching -see above for number of hatchlings analyzed. Not included in review: embryo and hatchling heart weight; embryo and hatchling liver weight; embryo and hatchling mortality; hatchability; embryo cardiac morphology; hatchling cardiac ultrasound; hatchling cardiac myofibril ATPase; hatchling serum PFOA concentration Notes Author responded to requests for additional information. Raw data provided by study author. Authors did not specify chemical form of PFOA (assume CAS# 335-67-1). In embryos, mortality was statistically significantly increased in 2 mg PFOA/kg egg group (76% increase compared to control). In hatchlings, mortality and hatching were statistically equal across doses and control groups.

Study Element Description Methods
Salmon endocrine and developmental toxicological study Participants Atlantic salmon (Salmo salar) Eggs obtained from supplier Total number of eggs allocated: Unclear Exposure Eggs exposed to PFOA in water through day 48. Hatching occurred at day 20. Larvae were collected at days 21, 35, 49, and 56. Exposure groups: • 1 dose groups = 100 µg PFOA/L water • 1 control group = water with carrier solvent (methanol) Outcomes 1. Length (cm) -larvae measured using microscope with ruler and digital camera. Number of larvae analyzed (same larvae used to examine dry weight outcome described below): • 10 randomly selected larvae for each time point and dose group 2. Dry weight (g) -larvae dried in heat cabinet and weighed using a micro-weight scale -see above for number of larvae analyzed. Not included in review: outcomes from treatment with PFOS; bone development; effects on HTP-axis; effects on ER expression; effects on GH-IGF axis; effects on chondrogenic and osteogenic pathways Notes Authors did not respond to requests for additional information. Authors did not specify chemical form of PFOA (assume CAS# 335-67-1). Data estimates for figures presented in the published paper were obtained using an online digital ruler. Study did not discuss survival.

Bias domain Authors' judgment Support for judgment Sequence generation
Probably high risk Randomization scheme not discussed; adequate randomization is not standard protocol for these types of experiments.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Low risk Based on author response, dams and offspring were adequately followed; author reported numbers allocated. Selective reporting Low risk Outcomes outlined in abstract/methods section of paper were reported in results section; "n" provided by author; raw data provided by author.

Conflict of interest
Probably low risk Associated funds and persons appear to be from government and/or academia only and free of financial interests in study results. However, no claim denying conflicts of interest was made.

Other bias
Low risk No other potential biases are suspected.   Hines et al. 2009 (study ID 260).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably low risk Mice were "randomly distributed among treatment groups", although details of random sequence generation were not discussed.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Probably low risk Based on author response, dams were adequately followed; offspring mortality not reported, but noted that losses equivalent between groups; author reported numbers allocated. Selective reporting Probably low risk Author reported different numbers allocated than reported in paper and noted that a figure in the paper displayed male and female weights even though the legend specifies female weights only; "n" provided by author; raw data provided by author.

Conflict of interest
Probably high risk Associated funds and persons appear to be from government and/or academia only and free of financial interests in study results. However, acknowledgement was made to employee from company financially invested in PFOA (Dow) for "constructive input on this manuscript" and no claim denying conflicts of interest was made. Other bias Low risk No other potential biases are suspected.  Fenton et al. 2009 (study ID 264).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably low risk Mice were "were weighed and randomly distributed among PFOA treatment groups", although details of random sequence generation were not discussed.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Probably high risk Based on author response, offspring mortality not assessed; dams were adequately followed; weighed one pup per litter; numbers allocated reported.

Selective reporting
Probably low risk Author reported that one pup per litter included in weight outcome, but this was not clearly stated in paper; "n" provided by author; raw data provided by author.

Conflict of interest
Low risk Funding source not stated, but associated persons appear to be from government and/or academia only and free of financial interests in study results. "The author declares that there are no conflicts of interest." Other bias Low risk No other potential biases are suspected.  White et al. 2009 (study ID 312).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably low risk "Upon arrival at the animal facility on GD 0, mice were weighed and randomly assigned to one of two treatment groups", although details of random sequence generation were not discussed.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Probably high risk Based on author response, offspring were adequately followed; only reported on results from 3 pups weighed from each of 4 dams (56 allocated/group); numbers allocated reported. Selective reporting Low risk Outcomes outlined in abstract/methods section of paper were reported in results section; adequate "n" reported; raw data provided by author.

Conflict of interest
Probably high risk Associated funds and persons appear to be from government and/or academia only and free of financial interests in study results. "The authors declare that there are no conflicts of interest." However, company financially invested in PFOA (3M) provided analysis of PFOA chemical used in study.

Other bias
Low risk No other potential biases are suspected.  Abbott et al. 2007 (study ID 528).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably low risk "Plug positive female mice were weighed, randomly assigned to treatment groups", although details of random sequence generation were not discussed.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Low risk Based on author response, dams and offspring were adequately followed; author reported numbers allocated. Selective reporting Low risk Outcomes outlined in abstract/methods section of paper were reported in results section; "n" provided by author; raw data provided by author.

Conflict of interest
Probably low risk Funding source not stated, but associated persons appear to be from government and/or academia only and free of financial interests in study results. However, no claim denying conflicts of interest was made.

Other bias
Low risk No other potential biases are suspected.  White et al. 2007 (study ID 566).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably low risk "Animals were weighed upon arrival and randomly distributed among four treatment groups", although details of random sequence generation were not discussed.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Low risk Based on author response, dams and offspring were adequately followed; numbers allocated reported. Selective reporting Low risk Outcomes outlined in abstract/methods section of paper were reported in results section; "n" provided by author; raw data provided by author.

Conflict of interest
Probably low risk Associated funds and persons appear to be from government and/or academia only and free of financial interests in study results. However, no claim denying conflicts of interest was made.

Other bias
Low risk No other potential biases are suspected.  Wolf et al. 2007 (study ID 571).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably low risk "Upon arrival at the animal facility, mice were weighed and randomly assigned", although details of random sequence generation were not discussed.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Low risk Dams and offspring were adequately followed; numbers allocated reported. Selective reporting Low risk Outcomes outlined in abstract/methods section of paper were reported in results section; "n" provided by author; raw data provided by author.

Conflict of interest
Probably high risk Funding source not stated, but associated persons appear to be from government and/or academia only and free of financial interests in study results. However, company financially invested in PFOA (3M) provided analysis of PFOA chemical used in study and no claim denying conflicts of interest was made.

Other bias
Low risk No other potential biases are suspected.  Lau et al. 2006 (study ID 635).

Bias domain Authors' judgment Support for judgment Sequence generation
High risk "Animals were randomly assigned to treatment groups". Author noted that mice were ranked according to weight at arrival then assigned evenly to each group at "random" but that a component such as a random number generator was not used in the sequence generation process.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Probably low risk Dams and offspring were adequately followed; numbers allocated not reported.

Selective reporting
Probably low risk Outcomes outlined in abstract/methods section of paper were reported in results section; adequate "n" provided by author for fetal and birth outcomes (pups per litter provided as range).

Conflict of interest
Probably high risk Associated funds and persons appear to be from government and/or academia only and free of financial interests in study results. However, company financially invested in PFOA (3M) provided analysis of PFOA chemical used in study and no claim denying conflicts of interest was made.

Other bias
Low risk No other potential biases are suspected.  Hinderliter et al. 2005 (study ID 711).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably low risk "Dams were ranked on…body weights and assigned to control and experimental groups by random sampling from the ranked list...Rats in each group were then randomly assigned to each subset", although details of random sequence generation were not discussed.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Low risk Based on author response, dams and pups were adequately followed; numbers allocated reported. Selective reporting Low risk Weight outcome outlined in methods section of paper, but not reported in results section; however, detailed outcome data presented in report provided by author; "n" provided by author; raw data provided by author.

Conflict of interest
High risk Du Pont and 3M sponsored study and authors were employed by companies.

Other bias
Low risk No other potential biases are suspected.  Staples et al. 1984(study ID 1871.

Bias domain Authors' judgment Support for judgment Sequence generation
High risk Females were ranked within breeding days by body weight and assigned to groups by rotating in order of rank. Allocation concealment High risk Females were allocated by rotation. Blinding Probably low risk "To limit possible bias in the examination of maternal and fetal specimens, the dams were coded (group designation unknown to examiner) from just before sacrifice until all maternal and fetal data were collected"; unclear if applies to birth outcomes. Incomplete outcome data Low risk Dams and offspring were adequately followed; numbers allocated reported.

Selective reporting
Probably low risk Outcomes outlined in abstract/methods section of paper were reported in results section; average values reported so can calculate "n" analyzed.

Conflict of interest
High risk Du Pont sponsored study and authors were employed by the company.

Other bias
Low risk No other potential biases are suspected.  Boberg et al. 2008 (study ID 3061).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably low risk "The dams were randomized into seven groups of eight with similar body weight distributions", although details of random sequence generation were not discussed.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Low risk Based on author response, dams and pups were adequately followed; numbers allocated reported. Selective reporting Probably low risk Author reported different numbers allocated than reported in paper; weight outcome pre-specified in methods section of paper and reported as "data not shown" in results section; however, detailed outcome data provided by author; "n" provided by author; raw data provided by author.

Conflict of interest
Low risk Associated funds and persons appear to be from government and/or academia only and free of financial interests in study results. Authors claim "none" for conflicts of interest.

Other bias
Low risk No other potential biases are suspected.  Onishchenko et al. 2011 (study ID 3610).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably high risk Randomization scheme not discussed; adequate randomization is not standard protocol for these types of experiments.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Probably high risk Dams and offspring were not adequately followed, but noted "litter size and sex ration were similar in control and exposed groups"; number allocated reported, but author provided data for more dams than allocated. Selective reporting Probably high risk Weight outcome reported in results section of paper, but not pre-specified in methods section; "n" provided by author, but unclear if pup number or dam number and greater than numbers allocated in paper; author provided raw data.

Conflict of interest
Probably low risk Associated funds and persons appear to be from government and/or academia only and free of financial interests in study results. However, no claim denying conflicts of interest was made.

Other bias
Low risk No other potential biases are suspected.  White et al. 2011 (study ID 3862).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably low risk "Timed pregnant dams were randomly distributed among five treatment groups", although details of random sequence generation were not discussed.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Low risk Dams and offspring were adequately followed; numbers allocated reported, but author provided different numbers allocated. Selective reporting Probably low risk Author reported different numbers allocated than reported in paper; weight outcome outlined in methods section paper, but not reported in results section; however, detailed outcome data provided by author; "n" provided by author; raw data provided by author.

Conflict of interest
Low risk Associated funds and persons appear to be from government and/or academia only and free of financial interests in study results. "The authors declare they have no actual or potential competing financial interests." Other bias Low risk No other potential biases are suspected.

Bias domain Authors' judgment Support for judgment Sequence generation
Low risk "Upon arrival parental generation rats will be assigned to individual housing on the basis on computer-generated random units…The rats will be assigned to dosage groups based on computer-generated (weight-ordered) randomization procedures." Allocation concealment Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Low risk Dams and offspring were adequately followed; numbers allocated reported. Selective reporting Low risk Outcomes outlined in abstract/methods section of paper were reported in results section; adequate "n" reported.

Conflict of interest
High risk 3M sponsored study and authors were employed by company.

Other bias
Low risk No other potential biases are suspected.  Hagenaars et al. 2011 (study ID 59).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably high risk Randomization scheme not discussed; adequate randomization is not standard protocol for these types of experiments.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Low risk Hatchability and mortality reported; numbers allocated reported. Selective reporting Probably low risk Author reported different numbers allocated than reported in paper; "n" provided by author; raw data provided by author.

Conflict of interest
Probably low risk Associated funds and persons appear to be from government and/or academia only and free of financial interests in study results. However, no claim denying conflicts of interest was made.

Other bias
Low risk No other potential biases are suspected.  Wang et al. 2010 (study ID 86).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably low risk "Cohorts of…flies were assigned randomly into vials"; unclear if applies to growth outcomes; details of random sequence generation were not discussed.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Probably high risk Hatchability and mortality not reported; only subset of treatment group analyzed for outcome; numbers allocated reported.

Selective reporting
Probably low risk Pupae weight outcome reported in results section of paper, but not pre-specified in methods section; "n" provided by author; raw data provided by author.

Conflict of interest
Probably low risk Associated funds and persons appear to be from government and/or academia only and free of financial interests in study results. However, no claim denying conflicts of interest was made.

Other bias
Low risk No other potential biases are suspected.  Pinkas et al. 2010 (study ID 187).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably high risk Randomization scheme not discussed; adequate randomization is not standard protocol for these types of experiments.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Probably low risk Hatchlings were adequately followed; numbers allocated not reported.

Selective reporting
Probably high risk Weight outcome reported in results section paper, but not prespecified in methods section; adequate "n" reported.

Conflict of interest
Low risk Associated funds and persons appear to be from government and/or academia only and free of financial interests in study results. "No author has any conflict of interest to disclose." Other bias Low risk No other potential biases are suspected.  'Brien et al. 2009 (study ID 236).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably low risk "Eggs were randomly distributed among 4 dose groups", although details of random sequence generation were not discussed.

Allocation concealment
Probably high risk Allocation concealment not discussed; adequate concealment is not standard protocol for these types of experiments. Blinding Probably high risk Blinding not discussed; adequate blinding is not standard protocol for these types of experiments. Incomplete outcome data Low risk Based on author response, embryos were adequately followed; numbers allocated reported. Selective reporting Low risk Weight outcome outlined in methods section paper, but not reported in results section; however, detailed outcome data provided by author; "n" provided by author; raw data provided by author.

Conflict of interest
Low risk Associated funds and persons appear to be from government and/or academia only and free of financial interests in study results. "The authors declare that there are no conflicts of interest." Other bias Low risk No other potential biases are suspected.  Jiang et al. 2012 (study ID 3926).

Bias domain Authors' judgment Support for judgment Sequence generation
Probably high risk "Eggs were...given ID numbers, and evenly distributed by weight among doses"; adequate randomization is not standard protocol for these types of experiments. Allocation concealment Probably low risk Eggs "given ID numbers" before allocation so there may have been allocation concealment, but not explicitly stated. Blinding Probably low risk Eggs were "given ID numbers" so blinding may have been applied, but not explicitly stated. Incomplete outcome data Probably high risk Hatching and mortality reported, but authors noted mortality not carefully tracked for all eggs included in outcome measurement; author reported numbers allocated. Selective reporting Probably low risk Weight and length outcomes reported in results section of paper, but not pre-specified in methods section; "n" provided by author; raw data provided by author.

Selective reporting
Probably low risk Weight and length outcomes reported in results section of paper, but not pre-specified in methods section; "n" provided by author; raw data provided by author.

Conflict of interest
Low risk Funding source not stated, but associated persons appear to be from government and/or academia only and free of financial interests in study results. "No conflicts of interest." Other bias Low risk No other potential biases are suspected.   Figure 5A).   Figure 5B). The confidence intervals are for the difference in means comparing each dose group to the control. These were calculated by us based on the information available to us (mean and standard error estimates). Asterisks indicate statistically significant (p<0.05) difference in response compared to control group, as reported by the study authors.
The information available to us differed from that available to the study authors, and statistical tests used varied by study; therefore there is not a one-to-one correspondence with the confidence intervals as calculated by us and the pvalues reported by the study authors. a mg/kg BW/day, unless otherwise specified. b Study split into 2 datasets; a) cross foster (exposure GD1-17); b) windows of sensitivity (exposure groups GD7-17, GD10-17, GD13-17, GD15-17),  Figure 6A).   Figure 6B). The confidence intervals are for the difference in means comparing each dose group to the control. These were calculated by us based on the information available to us (mean and standard error estimates). Asterisks indicate statistically significant (p<0.05) difference in response compared to control group, as reported by the study authors.
The information available to us differed from that available to the study authors, and statistical tests used varied by study; therefore there is not a one-to-one correspondence with the confidence intervals as calculated by us and the p-values reported by the study authors.
a Study did not test for statistical significance.

Sequence generation
Was the allocation sequence adequately generated?
Criteria for a judgment of 'YES' (i.e. low risk of bias): The investigators describe a random component in the sequence generation process such as: • Referring to a random number table; • Using a computer random number generator; • Coin tossing; • Shuffling cards or envelopes; • Throwing dice; • Drawing of lots.
Criteria for the judgment of 'PROBABLY YES' (i.e. probably low risk of bias): There is insufficient information about the sequence generation process to permit a judgment of 'YES', but there is indirect evidence that suggests the sequence generation process was random, as described by the criteria for a judgment of 'YES'.
Criteria for the judgment of 'PROBABLY NO' (i.e. probably high risk of bias): There is insufficient information about the sequence generation process to permit a judgment of 'NO', but there is indirect evidence that suggests a non-random component in the sequence generation process, as described by the criteria for a judgment of 'NO'.
Criteria for the judgment of 'NO' (i.e. high risk of bias): The investigators describe a non-random component in the sequence generation process or that a random component was not used. Usually, the description would involve some systematic, nonrandom approach, for example: • Sequence generated by date of birth; • Sequence generated by some rule based on date (or day) of arrival at facility; • Sequence generated by some rule based on record number. Other non-random approaches happen much less frequently than the systematic approaches mentioned above and tend to be obvious. They usually involve judgment or some method of nonrandom categorization of animals, for example: • Allocation by judgment of the investigator; • Allocation by availability of the intervention.
There is evidence that sequence generation is not an element of study design capable of introducing risk of bias in the study.

Allocation concealment
Was allocation adequately concealed?
Criteria for a judgment of 'YES' (i.e. low risk of bias): Investigators could not foresee assignment because one of the following, or an equivalent method, was used to conceal allocation: • Sequentially numbered treatment containers of identical appearance to control; or • Sequentially numbered prepared route of administration (e.g., pre-prepared water dosed with chemical) of identical appearance; or • Sequentially numbered, opaque, sealed envelopes.
Criteria for the judgment of 'PROBABLY YES' (i.e. probably low risk of bias): There is insufficient information about allocation concealment to permit a judgment of 'YES', but there is indirect evidence that suggests the allocation was adequately concealed, as described by the criteria for a judgment of 'YES'.
Criteria for the judgment of 'PROBABLY NO' (i.e. probably high risk of bias): There is insufficient information about allocation concealment to permit a judgment of 'NO', but there is indirect evidence that suggests the allocation was not adequately concealed, as described by the criteria for a judgment of 'NO'.
Criteria for the judgment of 'NO' (i.e. high risk of bias): Investigators handling experimental animals could possibly foresee assignments and thus introduce selection bias, such as allocation based on: • Using an open random allocation schedule (e.g. a list of random numbers); or • Assignment envelopes were used without appropriate safeguards (e.g. if envelopes were unsealed or non-opaque or not sequentially numbered); or • Alternation or rotation; or • Non-random and known criteria, such as date of birth; or • Record number; or • Any other explicitly unconcealed procedure.
There is evidence that allocation concealment is not an element of study design capable of introducing risk of bias in the study.

Blinding of personnel and outcome assessors
Was knowledge of the allocated interventions adequately prevented during the study?
Criteria for a judgment of 'YES' (i.e. low risk of bias): Any one of the following: • No blinding, but the review authors judge that the outcome and the outcome measurement are not likely to be influenced by lack of blinding; or • Blinding of key study personnel ensured, and unlikely that the blinding could have been broken; or • Some key study personnel were not blinded, but outcome assessment was blinded and the non-blinding of others unlikely to introduce bias.
Criteria for the judgment of 'PROBABLY YES' (i.e. probably low risk of bias): There is insufficient information about blinding to permit a judgment of 'YES', but there is indirect evidence that suggests the study was adequately blinded, as described by the criteria for a judgment of 'YES'.
Criteria for the judgment of 'PROBABLY NO' (i.e. probably high risk of bias): There is insufficient information about blinding to permit a judgment of 'NO', but there is indirect evidence that suggests the study was not adequately blinded, as described by the criteria for a judgment of 'NO'.
Criteria for the judgment of 'NO' (i.e. high risk of bias): Any one of the following: • No blinding or incomplete blinding, and the outcome or outcome measurement is likely to be influenced by lack of blinding; or • Blinding of key study personnel attempted, but likely that the blinding could have been broken; or • Some key study personnel were not blinded, and the non-blinding of others likely to introduce bias.
There is evidence that blinding is not an element of study design capable of introducing risk of bias in the study.

Incomplete outcome data
Were incomplete outcome data adequately addressed?
Criteria for a judgment of 'YES' (i.e. low risk of bias): The number of animals assessed for outcome of interest is reported and data is provided indicating adequate follow up of all treated animals. Additional information provided by authors should be considered when making risk of bias judgments about incomplete outcome data. Additionally, any one of the following: • No missing outcome data; or • Reasons for missing outcome data unlikely to be related to true outcome (for survival data, censoring unlikely to be introducing bias); or • Missing outcome data is provided and is balanced in numbers across intervention groups, with similar reasons for missing data across groups; or • For dichotomous outcome data, the proportion of missing outcomes compared with observed event risk not enough to have a biologically relevant impact on the intervention effect estimate; or • For continuous outcome data, plausible effect size (difference in means or standardized difference in means) among missing outcomes not enough to have a biologically relevant impact on observed effect size; or • Missing data have been imputed using appropriate statistical methods.
Criteria for the judgment of 'PROBABLY YES' (i.e. probably low risk of bias): There is insufficient information about incomplete outcome data to permit a judgment of 'YES', but there is indirect evidence that suggests incomplete outcome data were adequately addressed, as described by the criteria for a judgment of 'YES'.
Criteria for the judgment of 'PROBABLY NO' (i.e. probably high risk of bias): There is insufficient information about incomplete outcome data to permit a judgment of 'NO', but there is indirect evidence that suggests incomplete outcome data was not adequately addressed, as described by the criteria for a judgment of 'NO'.
The number of animals allocated not reported and no data is provided to indicate that there was adequate follow up of all treated animals. Additionally, any one of the following: • Reason for missing outcome data likely to be related to true outcome, with either imbalance in numbers or reasons for missing data across intervention groups; or • For dichotomous outcome data, the proportion of missing outcomes compared with observed event risk enough to induce biologically relevant bias in intervention effect estimate; or • For continuous outcome data, plausible effect size (difference in means or standardized difference in means) among missing outcomes enough to induce biologically relevant bias in observed effect size; or • 'As-treated' analysis done with substantial departure of the intervention received from that assigned at randomization; or • Potentially inappropriate application of simple imputation.
Criteria for the judgment of 'NOT APPLICABLE' (risk of bias domain is not applicable to study): There is evidence that incomplete outcome data is not capable of introducing risk of bias in the study.

Selective outcome reporting
Are reports of the study free of suggestion of selective outcome reporting?
Criteria for a judgment of 'YES' (i.e. low risk of bias): All of the study's pre-specified (primary and secondary) outcomes outlined in the methods, abstract, and/or introduction that are of interest in the review have been reported in the prespecified way, including the number of animals analyzed for outcomes of interest. Additional information provided by authors should be considered when making risk of bias judgments for selective outcome reporting.
Criteria for the judgment of 'PROBABLY YES' (i.e. probably low risk of bias): There is insufficient information about selective outcome reporting to permit a judgment of 'YES', but there is indirect evidence that suggests the study was free of selective reporting, as described by the criteria for a judgment of 'YES'.
Criteria for the judgment of 'PROBABLY NO' (i.e. probably high risk of bias): There is insufficient information about selective outcome reporting to permit a judgment of 'NO', but there is indirect evidence that suggests the study was not free of selective reporting, as described by the criteria for a judgment of 'NO'.
Criteria for the judgment of 'NO' (i.e. high risk of bias): The study was not free of selective reporting. The following should be considered: • Authors did not report numbers analyzed for outcomes of interest; or • Not all of the study's pre-specified primary outcomes (as outlined in the protocol, title, abstract, and/or introduction) that are of interest in the review have been reported; or • One or more primary outcomes is reported using measurements, analysis methods or subsets of the data (e.g. subscales) that were not pre-specified (unless clear justification for their reporting is provided, such as an unexpected adverse effect); or • One or more reported primary outcomes were not pre-specified (unless clear justification for their reporting is provided, such as an unexpected adverse effect); or • One or more outcomes of interest in the review are reported incompletely so that they cannot be entered in a meta-analysis; or • The study report fails to include results for a key outcome that would be expected to have been reported for such a study.
Criteria for the judgment of 'NOT APPLICABLE' (risk of bias domain is not applicable to study): There is evidence that selective outcome reporting is not capable of introducing risk of bias in the study.

Other potential threats to validity
Was the study apparently free of other problems that could put it at a risk of bias?
Criteria for a judgment of 'YES' (i.e. low risk of bias): The study appears to be free of other sources of bias.
Criteria for the judgment of 'PROBABLY YES' (i.e. probably low risk of bias): There is insufficient information to permit a judgment of 'YES', but there is indirect evidence that suggests the study was free of other threats to validity, as described by the criteria for a judgment of 'YES'.
The study did not receive support from a company, study author, or other entity having a financial interest in the outcome of the study. A conflict of interest statement is provided to indicate the authors have no financial interest and there is evidence of the entities not having a financial interest. Examples of this evidence include the following: • Funding source is limited to government, non-profit organizations, or academic grants funded by government, foundations and/or non-profit organizations; • Chemicals or other treatment used in study were purchased from a supplier; • Company affiliated staff are not mentioned in the acknowledgements section; • Authors were not employees of a company with a financial interest in the outcome of the study; • Company with a financial interest in the outcome of the study was not involved in the design, conduct, analysis, or reporting of the study and authors had complete access to the data; • Study authors make a claim denying conflicts of interest; • Study authors are unaffiliated with companies with financial interest, and there is no reason to believe a conflict of interest exists; • All study authors are affiliated with a government agency (are prohibited from involvement in projects for which there is a conflict of interest or an appearance of conflict of interest).
Criteria for the judgment of 'PROBABLY YES' (i.e. probably low risk of bias): There is insufficient information to permit a judgment of 'YES', for example there is no conflict of interest statement denying financial interests, but there is evidence that suggests the study was free of support from a company, study author, or other entity having a financial interest in the outcome of the study, as described by the criteria for a judgment of 'YES'.
Criteria for the judgment of 'PROBABLY NO' (i.e. probably high risk of bias): There is insufficient information to permit a judgment of 'NO', but there is indirect evidence that suggests the study was not free of support from a company, study author, or other entity having a financial interest in the outcome of the study, as described by the criteria for a judgment of 'NO'.
Criteria for the judgment of 'NO' (i.e. high risk of bias): The study received support from a company, study author, or other entity having a financial interest in the outcome of the study. Examples of support include: • Research funds; • Writing services; • Author/staff from study was employee or otherwise affiliated with company with financial interest; • Company limited author access to the data; • Company was involved in the design, conduct, analysis, or reporting of the study; • Study authors claim a conflict of interest.
Criteria for the judgment of 'NOT APPLICABLE' (risk of bias domain is not applicable to study): There is evidence that conflicts of interest are not capable of introducing risk of bias in the study.