Hexavalent chromium and stomach cancer: a systematic review and meta-analysis

Figures & data

Table 1. Inclusion criteria.

Category	Details
For inclusion	Experimental animals with chronic administration of Cr(VI) by drinking water or oral intake
	Humans with non-occupational (environmental) exposures to Cr(VI) via ingestion
	Humans with occupational exposures to Cr(VI) Workers in chromate production, stainless-steel welding, chrome pigment production, chrome plating/electroplating, ferrochrome production industries Leather tanners included if the authors of primary studies specifically indicate exposure to Cr(VI) or process such as “two bath” process Cement workers included if authors of primary studies indicated that the occupation involved cement production because Cr(VI) is known to potentially produced in the kiln
	Other occupations (not specified above) will be included if the authors of primary studies indicated that workers were exposed to Cr(VI). This distinction was made because of evidence of increased cancer risk from Cr(VI) exposure has not been specifically reported for these other occupations generally, and other risk factors including asbestos, solvent, and arsenic exposures, have been indicated.
For exclusion	Workers with no occupational Cr(VI) exposure. Individuals with no non-occupational (environmental) Cr(VI) exposure. Human occupational and non-occupational studies, where exposure to Cr(VI) is not specifically evaluated nor stated. Experimental animals without chronic ingestion of Cr(VI).
	Studies that do not provide quantitative data of stomach cancer risk morbidity or mortality, or risks cannot be reasonably calculated based on the information provided by the authors of the primary studies
	Proportionate mortality ratio (PMR) and proportionate cancer mortality ratio (PCMR) studies Studies that do not assess risk at the individual level (e.g. registry studies based on occupational titles, ecological studies)

Table 2. Meta-analysis descriptions.

Analysis	Description
1	All Studies
2*	From Analysis 1, eliminate Tier 3 studies with -2 or -1 ratings in the key questions (Approach 1 for tier classification)^a^,c
3*	Tier 1 studies only – Based on overall risk of bias ratings (Approach 2 for tier classification) ^b,c
4a4b	Cohort studies only Case-control studies only^d
5	Sub-groups: Identified by the study authors as high exposure groups^e
6	Industries associated with increased lung cancer risk from Cr(VI) (i.e., chromate production, pigment production, plating, ferrochromium production)
7*	From Analysis 4a, eliminate Tier 3 studies (Approach 1 for tier classification)^a^,c
8*	From Analysis 6, eliminate Tier 3 studies (Approach 1 for tier classification)^a^,c

*Integration of the risk of bias assessment results for the meta-analysis.

^a Approach 1 for tier classification: Greater emphasis is placed on the ratings for the key questions as identified by NTP OHAT (2015). There were 31 studies classified as Tier 3.

^b Approach 2 for tier classification: overall average ratings for the risk of bias questions is considered for tiering.

^c No Tier 3 studies were identified with Approach 2. Approach 1 only identified one study for Tier 1 (Iaia et al. 2006).

^d One case-control study was classified as Tier 3 (Moulin et al. 1992), which only left two studies (Krstev et al. 2005; Xu et al. 1996). Thus, additional analyses for the case-control study category was not conducted.

^e All studies for Analysis 5 were classified as Tier 3 studies in the risk of bias assessment (Approach 1 for tier classification). Thus, additional analyses for the high exposure subcohort category were not conducted.

Figure 1. Literature Search Results (in accordance to PRISMA reporting). *Hand searching was based on the reference citations of previously published meta-analyses and recent government documents, i.e., ATSDR 2012; Cole and Rodu 2005; Gatto et al. 2010; NIOSH 2013; Welling et al. 2015.

Table 3. Data extraction of experimental animal studies (n = 4).

Study	Species (strain)	Gender (number)	Test compound and dose groups	Route, Cr(VI) concentration/dose^a	Study duration	Stomach tumor incidence	Overall tier for risk of bias^b	Limitations (as stated by author)
NTP (2008)	Rat, F344/N	Males (50 per dose group), females (50 per dose group)	Cr(VI) as sodium dichromate dihydrate (4 dose groups per gender per species) Negative control group also included per gender per species (tap water)	Ingestion (drinking water) Concentration: 0, 5, 20, 60, 180 mg/L (M/F) Dose: 0, 0.21, 0.77, 2.1, 5.9 mg/kg/day (M); 0, 0.24, 0.94, 2.4, 7.0 mg/kg/day (F)	2 years	Male: forestomach - 0/50, 0/50, 0/49, 0/50, 0/49; glandular stomach - 1/50,0/50, 0/49, 0/50, 1/49 Female: forestomach - 0/50, 0/50, 0/50, 0/50, 0/50; glandular stomach - 0/50, 0/50, 0/50, 0/50, 0/50 No evidence of Cr(VI)-related stomach cancer	1	None stated
NTP (2008)	Mouse, B6C3F1	Males (50 per dose group), females (50 per dose group)		Ingestion (drinking water) Concentration: 0, 5, 20, 60, 180 mg/L (F); 0, 5, 10, 30, 90 mg/L (M) Dose: 0, 0.38, 0.91, 2.4, 5.9 mg/kg/day (M); 0, 0.38, 1.4, 3.1, 8.7 mg/kg/day (F)	2 years	Male: forestomach - 1/50, 0/50, 0/48, 4/50, 0/48; glandular stomach - 1/50, 0/48, 0/48, 2/50, 0/47 Female: forestomach - 1/49, 2/49, 0/50, 0/48, 3/50; glandular stomach - 0/49, 1/48, 0/50, 0/48,0/50 No evidence of Cr(VI)-related stomach cancer	1	None stated
Borneff et al. (1968)	Mouse, White NMRI	Males (40), females (480)	4 groups. Group 1: detergent, Group 2: detergent plus Cr(VI) as potassium chromate, Group 3: detergent plus benzo[a]pyrene, Group 4: benzo[a]pyrene plus Cr(VI) as potassium chromate	Ingestion (drinking water) Household detergent "Pril" added to drinking water: 3% Benzo[a]pyrene (Group 4): 10 μg/ml (range 7-15) Cr(VI): 134 mg/L chosen as max tolerated without developing any damage; remained constant during the first 3 days. Was 80 mg/L after 3 weeks. Average uptake in Group 2: 13.5 mg/kg/day; Group 4: 12 mg/kg/day	3-generation (880 days)	Group 2: 2/66 F developed forestomach carcinoma; 9/66 F, 1/35 M developed forestomach papilloma. Group 4: 23/80 F, 12/26 M developed forestomach carcinoma. 26/80 F, 4/26 M developed forestomach papilloma. Vehicle controls also developed forestomach papilloma (F, 2/79; M, 3/47) but not carcinoma. The incidence of forestomach tumors in treated mice was not significantly higher than controls; study authors concluded evidence of carcinogenicity was equivocal.	3	Authors mentioned that groups that were given Cr(VI) (especially Group 4), cannibalism occurred. It was also mentioned that mice only reluctantly accepted the mixture of detergent, benzo[a]pyrene, and Cr(VI) in their drinking water (taking up a fluid closer to their requirements). Continuous decrease in the average weights from 7 month and onward observed for Group 4. In the 8th month of the experiment, an ectromely epidemy occurred in mice. Within 3 months, 512 animals died.
Mackenzie et al. (1958)	Rat, Sprague-Dawley albino	Experiment 1: 8 males and 8 females except the control group which contained 10 rats of each sex. Experiment 2: 9 females and 12 males	Experiment 1: control group given distilled water, Groups 2 to 6 given Cr(VI) as potassium chromate Experiment 2: Group 7 (control) given distilled water, Group 8 given Cr(VI) as potassium chromate, Group 9 given Cr(III) as chromic chloride	Ingestion (drinking water) Groups 2 to 6 in experiment 1: 0.12, 0.58. 1.2, 2.1, 3.0 mg/L Group 8 in experiment 2: 6.7 mg/L	1 year	No stomach tumors were reported	2	Mortality from respiratory infection occurred in both experiments.

M: Males; F: Females.

^aConcentrations/doses were converted based on molecular weight of Cr(VI) and molecular weight of the test compound. For Borneff et al. (1968), average bodyweight of 0.02 kg assumed for mice in calculation of doses.

^bBased on Approach 1 for tier classification.

Figure 2. Risk of bias assessment and tier classifications for experimental animal studies (n = 4). Colors: red, (−−; definitely high risk of bias), pink (−; probably high risk of bias), light green (+; probably low risk of bias), dark green (++; definitely low risk of bias). Tiers: 1 (Tier 1), 2 (Tier 2), 3 (Tier 3).

Table 4. Summary of evidence synthesis and confidence in experimental animal studies.

Study type	Number of studies	Findings	Initial confidence rating^a	Risk of bias	Unexplained inconsistency	Indirectness	Imprecision	Magnitude of effect	Dose-response	Consistency across study types	Final confidence rating^b
Oral ingestion studies	4	No increases in stomach cancer incidence, not statistically significant, or no stomach cancers observed	High	– Two studies Tier 1 ↑; single Tier 3 ↓	– No unexplained inconsistencies	– All studies designed to directly evaluate oral exposures to Cr(VI) and cancer	– No imprecision reported in the 4 studies (Findings were negative, increasing confidence that no imprecision observed in any study)	↑ No significant effect observed in the 4 studies (increases confidence in no/low magnitude of effect)	– No dose-response reported in the 4 studies (increases confidence that no dose-response observed in any study)	↑ Results are consistent; no significantly increased stomach cancer observed in the 4 studies	High (++++): high confidence in the lack of effect

– No change in the initial confidence rating.

↑ Upgrade in the initial confidence rating.

↓ Downgrade in the initial confidence rating.

^aBased on NTP OHAT (2015) Table 8 – Study design features for initial confidence rating.

^bAccording to NTP OHAT (2015) Figure 6, factors decreasing confidence include risk of bias, unexplained inconsistency, indirectness, and imprecision. Factors increasing confidence include magnitude of effect, dose response, and consistency across study types.

Figure 3. Funnel plot of all occupational studies (n = 44) included for meta-analysis.

Figure 4. Meta-analysis 1: All human occupational studies (n = 44). For the individual studies, 95%CIs are calculated intervals based on the effect size and parameters of the random-effects model.

Figure 5. Meta-analysis 2: Tier 3 removed from human occupational studies (remaining studies, n = 13). For the individual studies, 95%CIs are calculated intervals based on the effect size and parameters of the random-effects model. Tier 3: Studies with definitely high or probably high risk of bias for at least two of the key NTP OHAT questions (Q4, Q8, and Q9 related to exposure, confounding, and outcome, respectively).

Table 5. Meta-analysis results (Human occupational data).

Analysis	Description	Studies (N)	Meta-RR	95% CI	I^2
1	All Studies	44	1.08	0.96 to 1.21	25.1
2*	Eliminate Tier 3 studies from Analysis 1 (Approach 1 for tier classification)^a^,c	13	1.03	0.84 to 1.26	19.8
3*	Tier 1 studies only – Based on overall ratings (Approach 2 for tier classification)^b,c	8	0.90	0.74 to 1.10	0.0013
4a	Cohort studies only	41	1.05	0.94 to 1.19	26.2
4b	Case-control studies only^d	3	1.82	0.98 to 3.40	33.8
5	Sub-groups: Identified by the study authors as high exposure groups^e	13	1.30	1.00 to 1.67	29.5
6*	Industries with evidence of increased lung cancer risk^f	19	1.11	0.90 to 1.37	28.8
7*	Eliminate Tier 3 studies from Analysis 4a (Approach 1 for tier classification)^a^,c	11	0.94	0.78 to 1.13	3.71
8*	Eliminate Tier 3 studies from Analysis 6 (Approach 1 for tier classification)^a^,c	7	1.14	0.77 to 1.68	34.9

*Integration of the risk of bias assessment results for the meta-analysis.

^a Approach 1 for tier classification: Greater emphasis is placed on the ratings for the key questions as identified by NTP OHAT. There were 31 studies classified as Tier 3.

^b Approach 2 for tier classification: The overall average ratings for the risk of bias questions is considered for tieri ng.

^c No Tier 3 studies were identified with Approach 2. Approach 1 only identified one study for Tier 1 (Iaia et al. 2006).

^d One case-control study was classified as Tier 3 (Moulin et al. 1992), which only left two studies (Krstev et al. 2005; Xu et al. 1996). Thus, additional analyses for the case-control study category was not conducted. All studies for Analysis 5 were classified as Tier 3 studies.

^e All studies for Analysis 5 were classified as Tier 3 studies in the risk of bias assessment (Approach 1 for tier classification). Thus, additional analyses for the high exposure subcohort category was not conducted.

^f Chromate production, pigment production, plating, and ferrochromium production are industries in which Cr(VI) exposure specifically has been shown to be associated with increased cancer risk.

Table 6. Summary of evidence synthesis and confidence in human occupational studies.

Study type^a	Number of studies^a	Findings^b	Initial confidence rating^c	Risk of bias	Unexplained inconsistency	Indirectness	Imprecision	Magnitude of effect	Dose-response	Consistency across study types	Final confidence rating^d
All studies	44	Not statistically elevated meta-RR	Low to moderate	↓ Probably or definitely high risk of bias in exposure and confounding domains	– Meta-analyses resulted in similar findings, including sub-analyses that assessed potential influence of study quality or size One study (Sorahan et al.1994) reported significantly elevated stomach cancer risk, but this Tier 3 study was of foundry workers with high risk of bias in exposure and confounding domains	– Studies were fit-for-purpose, evaluating disease mortality risk (including stomach cancer) from Cr(VI) exposure	– Confidence intervals around meta-RR are narrow	↑ Magnitude of effect was not large and not statistically elevated	NR Data are not available for most studies. For one study with exposure estimates, unable to establish dose-response in Proctor et al. (2016)	↑ Results are consistent across the meta-analyses (results are not statistically elevated). All individual studies (except Sorahan et al.1994) show consistency in the lack of statistically elevated risk of stomach cancer	Low (++) to moderate (+++) confidence in the human database
Cohort	41	Not statistically elevated meta-RR	Low to moderate
Case-control	3	Not statistically elevated meta-RR	Low to very low
Sub groups: Identified by study authors as high exposure groups	13	Not statistically elevated meta-RR	Low to moderate
Industries associated with increased lung cancer risk from Cr(VI)	19	Not statistically elevated meta-RR	Low to moderate

NR: Not reported.

– No change in the initial confidence rating.

↑ Upgrade in the initial confidence rating.

↓ Downgrade in the initial confidence rating.

^aBased on meta-analysis descriptions listed in Table 2.

^bBased on Meta-RRs presented in Table 5.

^cBased on NTP OHAT (2015) Table 8 – Study design features for initial confidence rating.

^dAccording to NTP OHAT (2015) Figure 6, factors decreasing confidence include risk of bias, unexplained inconsistency, indirectness, and imprecision. Factors increasing confidence include large magnitude of effect, dose response, and consistency across study types.

Figure 6. Application of the NTP OHAT (2015) framework of systematic review and evidence integration for developing hazard identification conclusions.

Table

SAB Member Name	Expertise	Roles and Responsibilities
Michael Goodman Loren Lipworth Tasha Beretvas#	Epidemiology Epidemiology Statistics	Overall: Provide guidance on methods for systematic review and meta-analysis of Cr(VI) exposure and stomach cancer risk. Specific tasks are: Discuss and agree upon the scope and focus of topic.  Approve systematic review framework (i.e. PRISMA) to be used. The framework to be used will be decided by ToxStrategies researchers. Review and approve draft protocol to be written by ToxStrategies researchers. The protocol will include research questions, key search terms, inclusion/exclusion criteria, literature search strategy, risk of bias assessment methods, and strategies for meta-analyses and analyses of subgroups. Review evidence tables (or summary of information) generated by ToxStrategies. Review meta-analyses results. Reviewed evidence synthesis and integration qualitative analysis results. Review and comment on manuscript drafts; serve as coauthor if desired^#

Table

Collaborator Name	Activities
Tasha Beretvas	SAB member Provided input to the protocol Directed statistical analyses to be performed Reviewed data extraction, risk of bias assessment, and meta-analysis results
Alea Goodmanson	Participated in data extraction for epidemiology studies Translated three studies in French to English to facilitate data extraction and risk of bias (Moulin et al. 1992; Moulin, Wild, Toamain, et al. 1993; Moulin et al. 1995)
Rick Nelson	Reviewed the manuscript draft to provide editorial comments
Annette Rohr	Organized the SAB Reviewed the manuscript draft and suggested wording changes for clarity of presentation

Table

Author Name	Activities
Mina Suh	Developed the protocol Reviewed literature search and screening results Conducted data extraction and risk of bias assessment and performed quality assurance/quality control Conducted evidence synthesis and integration qualitative analyses Designed and reviewed meta-analysis results Performed quality assurance/quality control of meta-analysis results Developed the initial manuscript draft, revised the manuscript, and finalized the manuscript
Daniele Wikoff	Developed the protocol Reviewed data extraction, risk of bias assessment, and meta-analysis results Reviewed evidence synthesis and integration qualitative analysis results Developed the initial manuscript draft, revised the manuscript, reviewed the final draft
Loren Lipworth	SAB Member Provided input to the protocol Reviewed data extraction, risk of bias assessment, and meta-analysis results Reviewed evidence synthesis and integration qualitative analysis results Reviewed and provided input on the manuscript (initial draft): development of introduction, study methods, results, and discussion sections Reviewed the revised draft and final manuscript
Michael Goodman	SAB Member Provided input to the protocol Reviewed data extraction, risk of bias assessment, and meta-analysis results Reviewed evidence synthesis and integration qualitative analysis results Reviewed and provided input on the manuscript (initial draft): development of introduction, study methods, results, and discussion sections Reviewed the revised draft and final manuscript
Seneca Fitch	Conducted literature search and screening Conducted data extraction and risk of bias assessment and performed quality assurance/quality control Reviewed the initial draft and final manuscript and provided edits
Liz Mittal	Conducted meta-analyses and subgroup analyses Developed funnel and forest plots in R Reviewed the final manuscript and provided edits
Caroline Ring	Provided oversight of meta-analyses Performed quality assurance/quality control of meta-analysis results Conducted Trim and Fill test Conducted Egger’s regression test Reviewed the final manuscript and provided edits
Deborah Proctor	Developed the protocol Reviewed literature search and screening results Reviewed data extraction, risk of bias assessment, and meta-analysis results Reviewed evidence synthesis and integration qualitative analyses Developed the manuscript draft, revised the manuscript, and finalized the manuscript

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