Using mechanistic information to support evidence integration and synthesis: a case study with inhaled formaldehyde and leukemia

Figures & data

Figure 1. Representation of the reaction of inhaled formaldehyde in mammalian nasal epithelium which rapidly reacts with macromolecules in the tissue and the albumin in the mucus that lines the respiratory epithelium resulting in a steep concentration gradient. After crossing the basement membrane, formaldehyde can react further with macromolecules in the submucosal layer or reach the systemic circulation. The nasal-associated lymphoid tissue (NALT) that is generally present near the ethmoid turbinates on either side of the nasal septum and near the ventral nasopharyngeal duct is one putative site of formaldehyde interactions with lymphoid tissues, but direct evidence that supports this hypothesis is lacking. (Figure reproduced from National Research Council (2011)).

Table 1. Problem formulation review questions.

1.Does inhalation exposure to exogenous formaldehyde add to increased amounts of endogenous formaldehyde at sites distant from the portal of entry such as the circulatory system? If so, were the analytical techniques utilized specific for exogenous formaldehyde?

2.Does inhalation exposure to formaldehyde lead to binding of exogenous formaldehyde to macromolecules in primary cells of the bone marrow and/or blood? If so, did the analytical techniques utilized measure specifically for the binding of exogenous formaldehyde?

3.Does inhalation exposure to formaldehyde lead to genotoxicity in primary cells at sites distant from the portal of entry? If so, did the analytical techniques utilized measure specifically for the effects of exogenous formaldehyde?

4.Based on the integration of the different streams of evidence (i.e. epidemiological, in vivo animal and mechanistic) are the postulated MOAs for increased risk of leukemia after inhalation of formaldehyde biologically plausible?

Table 2. Literature search strings*.

Chemical keyword		Exposure route keywords		Endpoint keyword		Excluded keyword
Formaldehyde	AND	(inhalation OR in vitro OR occupational OR environmental)	AND	Carcinogenicity	NOT	Oral
				Genotoxicity
				Mutagenicity
				macromolecular binding
				Crosslinks
				Leukemia
				Leukemogen
				Hematopoietic
				Cytotoxicity
				Pharmacokinetic
				Toxicokinetic
				Dosimetry
				Absorption
				Distribution
				Elimination
				Mechanism
				“Mode of action”
				Adduct
				Hematotoxicity

*All searches within PubMed included Medical Subject Headings (MeSH®), a comprehensive controlled vocabulary for indexing journal articles and books in the life sciences. MeSH® terms serve as a thesaurus that facilitates searching.

Table 3. Definitions of confidence levels and corresponding scores for individual study components.

Confidence level	Score	Definition
High	1	No notable deficiencies or concerns are identified in the domain metric that are likely to influence results.
Medium	2	Minor uncertainties or limitations are noted in the domain metric that are unlikely to have a substantial impact on results.
Low	3	Deficiencies or concerns are noted in the domain metric that are likely to have a substantial impact on results.
Unacceptable	4	Serious flaws are noted in the domain metric that consequently make the data/information source unusable
Not Applicable	NA	Rating of this metric is not applicable to the data source being evaluated. Therefore, no score is given.

Table 4. Definition of overall study quality levels and corresponding study quality scores.

Overall quality level	Definition	Overall quality score
High	No notable deficiencies or concerns are identified, and the data therefore could be used in the assessment with a high degree of confidence.	≥1 and <1.7
Medium	Possible deficiencies or concerns are noted, and the data therefore could be used in the assessment with a medium degree of confidence.	≥1.7 and <2.3
Low	Deficiencies or concerns are noted, and the data therefore could be used in the assessment with a low degree of confidence.	≥2.3 and ≤3
Unacceptable	Serious flaw(s) are identified and therefore, the data cannot be used for the assessment.	4

Table 5. SciRAP criterion for evaluating study relevance.

SciRAP criterion*	Considerations for relevance
In vivo studies
The identity of the test substance	Directly relevant – the study addresses the substance or agent (stressor) of interest for the hazard or risk assessment being conducted.
	Indirectly relevant – the study addresses a related substance or agent (stressor to the one of direct interest for the hazard or risk assessment being conducted.
	Not relevant – the study addresses a substance or agent (stressor) that is not relevant for the specific hazard or risk assessment being considered.
The animal model used	Directly relevant – there is no evidence that the animal model is irrelevant for the hazard or risk assessment being conducted.
	Indirectly relevant – there is no clear evidence that the animal model is irrelevant for the hazard or risk assessment being conducted. However, there may be a suspicion of species and/or strain differences affecting the sensitivity of the model.
	Not relevant – there is evidence that the animal model is not relevant for the hazard or risk assessment being conducted.
The endpoint studied	Directly relevant – the study addresses the endpoint of interest for the hazard or risk assessment being conducted.
	Indirectly relevant – the study addresses a related endpoint to the one of direct interest for the hazard or risk assessment being conducted.
	Not relevant – the study addresses an endpoint that is not relevant for the specific hazard or risk assessment being conducted
The route of administration	Directly relevant – the route of administration is the same (e.g. oral) as the route of exposure of interest for the hazard or risk assessment being conducted.
	Indirectly relevant – the route of administration is not the same as the route of exposure of interest for the hazard or risk assessment being conducted but the kinetics of the substance are sufficiently known to support extrapolations between different exposure routes.
The route of administration	Not relevant – the route of administration is not the same as the route of exposure of interest for the hazard or risk assessment being conducted and the kinetics of the substance are not sufficiently known to support extrapolations between different exposure routes. Or knowledge of the kinetics do not support extrapolations between different exposure routes.
The dose levels and resulting tissue levels.	Directly relevant – the study addresses dose and/or tissue levels of interest for predicted human exposure in the hazard or risk assessment being conducted.
	Indirectly relevant – the study addresses dose and/or tissue levels that are not directly relevant for predicted human exposure in the hazard or risk assessment being conducted but the characteristics of the dose-response are sufficiently known to support extrapolations.
	Not relevant – the dose and/or tissue levels addressed in the study are clearly not relevant for predicted human exposure in the hazard or risk assessment being conducted. Knowledge about the characteristics of the dose-response are do not support extrapolations between the doses tested and human exposure levels.
In vitro studies
The identity of the tested substance	Directly relevant – the study addresses the substance or agent (stressor) of interest for the hazard or risk assessment being conducted.
	Indirectly relevant – the study addresses a related substance or agent (stressor) to the one of direct interest for the hazard or risk assessment being conducted.
	Not relevant – the study addresses a substance or agent (stressor) that is not relevant for the specific hazard or risk assessment being conducted
The test system used	Directly relevant – the test system (cell line / cells / tissue / organ /embryo) is a relevant model for measurement of human health outcomes or modes of action/key events related to human health outcomes for the hazard or risk assessment being conducted.
	Indirectly relevant – the test system (cell line / cells / tissue / organ /embryo) is partly a relevant model for measurement of human health outcomes or modes of action/key events related to human health outcomes for the hazard or risk assessment being conducted. However, there are, for example, species or cell type differences reducing the relevance of the model.
The test system used	Not relevant – the test system (cell line / cells / tissue / organ /embryo) is not a relevant model for measurement of human health outcomes or modes of action/key events related to human health outcomes for the hazard or risk assessment being conducted.
The endpoint studied	Directly relevant – the study addresses the endpoint of interest for the human health outcomes or modes of action/key events related to the hazard or risk assessment being conducted.
	Indirectly relevant – the study addresses a related endpoint to the one of direct interest for the human health outcomes or modes of action/key events related to the hazard or risk assessment being conducted.
	Not relevant – the study addresses an endpoint that is not relevant for the human health outcomes or modes of action/key events related to the specific hazard or risk assessment being conducted.
The concentrations used	Directly relevant – the administered concentration levels are relevant for measured or predicted human exposure in the hazard or risk assessment being conducted.
	Indirectly relevant – the administered concentration levels are not directly relevant for measured or predicted human exposure in the hazard or risk assessment being conducted but the characteristics of the dose-response are sufficiently known to support extrapolations.
	Not relevant – the administered concentration levels are clearly not relevant for measured or predicted human exposure in the hazard or risk assessment being conducted. Knowledge about the characteristics of the dose-response does not support extrapolations between the concentrations tested and human exposure levels.

* http://www.scirap.org/Page/Index/a0130706-adce-45e0-83aa-64516c855fda/evaluate-reliabilityrelevance#

Table 6. Study quality and study relevance scoring results.

Study reference	Study type (in vivo, in vitro, or human)	Study quality score	Study relevance score
Casanova and Heck 1987	In vivo	1.5	Directly relevant
Casanova-Schmitz, Starr, et al. 1984	In vivo	1.5	Directly relevant
Dallas et al. 1992	In vivo	1.6	Directly relevant
Edrissi et al. 2013	In vivo	1.6	Directly relevant
Heck et al. 1989	In vivo	1.6	Directly relevant
Kleinnijenhuis et al. 2013	In vivo	1.2	Directly relevant
Kligerman et al. 1984	In vivo	1.6	Directly relevant
Lai et al. 2016	In vivo	1.2	Directly relevant
Leng et al. 2019	In vivo	1.3	Directly relevant
Lu et al. 2010	In vivo	1.6	Directly relevant
Lu et al. 2011	In vivo	1.3	Directly relevant
Meng et al. 2010	In vivo	1.1	Directly Relevant
Moeller et al. 2011	In vivo	1.4	Directly relevant
Neuss et al. 2010	In vitro	1.1	Directly relevant
Priha et al. 1996	In vitro	1.8	Directly relevant
Schmid and Speit 2007	In vitro	1.1	Directly relevant
Speit et al. 2009	In vivo	1.6	Directly relevant
Wei et al. 2017	In vivo	1.6	Not relevant*
Ye et al. 2013	In vivo	1.6	Not relevant*
Yu R et al. 2015	In vivo	1.4	Directly relevant
Zeller, Neuss, et al. 2011	Human	1.1	Directly relevant
Zhang et al. 2013	In vivo	1.5	Not relevant*

*These studies were considered not relevant to the problem formulation based on the use of methodologies that did not differentiate between exogenous and endogenous formaldehyde effects and/or the potential of impurities with the test substance due to the use of formalin (i.e. a mixture of water, methanol, and formaldehyde) as the test article.

Table 7. List of unacceptable studies determined during study quality evaluation.

Reference	Justification	Study quality score*
Casanova et al. 1988 (In vivo)	No source or purity of test substance used in the study was reported by study authors.	4
Fleig et al. 1982 (In vivo)	The study authors did not report methods for analyzing or preventing contamination of their blood samples.	4
Heck et al. 1985 (In vivo)	No source or purity of test substance used in the study was reported by study authors.	4
Ji et al. 2014 (In vitro)	The test substance purity was not reported. The test substance contained methanol. This study measures a biomarker of effect (i.e. aneuploidy, specifically monosomy 7 and trisomy 8 and micronuclei formation) that is not specific to the health outcome of leukemia.	4
Katsnelson et al. 2013 (In vivo)	The test substance was not properly identified, and the CAS number or test substance purity or source was not reported.	4
Liu et al. 2003 (Translated) (In vivo)	The test substance identity, form, source and purity could not be determined based on the information reported by study authors. The study authors report the test substance was generated from real wood-based panels loaded into the WH-2 small environment and climate chamber.	4
Speit et al. 2011 (In vitro)	This study measures a biomarker of effect (i.e. micronucleus formation) that is not specific to the health outcome of leukemia or its postulated MOA.	4
Viegas et al. 2010 (In vivo)	This study measures a biomarker of effect (i.e. micronucleus formation) that is not specific to the health outcome of leukemia or its postulated MOA.	4
Yu et al. 2014 (In vivo)	Information on the storage and preparation or methods of the test substance were not reported by study authors.	4
Yu G et al. 2015 (In vivo)	Information on the storage and preparation or methods of the test substance were not reported by study authors.	4
Zeller, Ulrich, et al. 2011 (In vitro)	The test substance identity, form and purity could not be determined. No source was reported for formaldehyde.	4
Zhang et al. 2010 (Human)	The study authors did not follow their own protocol. There were inconsistencies in the execution of study protocols. This study measures a biomarker of effect (i.e. aneuploidy, specifically monosomy 7 and trisomy 8) that is not specific to the health outcome of leukemia.	4
Zhao et al. 2004 (Translated) (In vivo)	The test substance identity, form, source and purity could not be determined based on the information reported by study authors. The study authors report the test substance was generated from real wood-based panels loaded into the WH-2 small environment and climate chamber.	4
Zhao et al. 2020 (In vivo)	The study authors did not report the number of animals per study group.	4
Zhao et al. 2020 (Ex vivo)	The study authors did not report the number of animals per study group.	4

*Three independent reviewers discussed and agreed on the metric score of 4.

Table 8. Overview of postulated MOAs.

MOA key events:	MOA 1: initiation of leukemia by direct damage to hematopoietic stem cells in bone marrow	MOA 2: toxicity to circulating blood stem cells and progenitors	MOA 3: targeting pluripotent nasal or oral cells	MOA 4: targeting blood stem cells and progenitors in the lung tissue
Event 1:	Conversion of formaldehyde to methanediol for transport through portal of entry	NP	NP	NP
Event 2:	Systemic delivery and transport of formaldehyde through blood	Systemic delivery and transport of formaldehyde through blood	NP	NP
Event 3:	Macromolecular binding to hematopoietic stem cells and progenitor blood cells residing in the bone marrow	Macromolecular binding to hematopoietic stem cells and progenitor blood cells in the circulatory system	NP	NP
Event 4:	Formaldehyde induced genotoxicity in the hematopoietic stem cells and progenitor blood cells derived from bone marrow or residing in the bone marrow	Formaldehyde induced genotoxicity in the hematopoietic stem cells and progenitor blood cells derived from bone marrow or residing in the bone marrow	Formaldehyde induced genotoxicity in primitive pluripotent cells in the nasal or oral tissue	Formaldehyde induced genotoxicity in hematopoietic stem cells and progenitor blood cells present in lung tissue (extravascular)
Event 5:	NP	NP	Damaged cell release into systemic circulation	Damaged stem cell release into systemic circulation
Event 6:	NP	Incorporation of initiated stem cell into the bone marrow	Incorporation of initiated pluripotent cell into the bone marrow	Incorporation of initiated stem cell into the bone marrow
Adverse outcome:	Initiation of Leukemia	Initiation of Leukemia	Initiation of Leukemia	Initiation of Leukemia

NP: not postulated.

Table 9. Summary of available studies associated with key events 2 and 3 of the postulated MOAs.

References	Species/strain/sex	Dose groups	Tissue assessed	Study quality (high, medium, low)	Results
Casanova and Heck 1987 (DNA Protein crosslink)	Rat/Fischer 344/M	0.9, 2, 4, 6, 10 ppm	Nasal tissue Respiratory tissue, Bone Marrow	High	Covalent macromolecular binding was determined using a dual-isotope method ([^3H]CH2O and [^14]CH2O). DNA protein crosslinking was significantly increased in the respiratory mucosa of rats following exposure to all concentrations of formaldehyde tested. However, macromolecular binding was not reported in the bone marrow following inhalation of any concentration of formaldehyde.
Casanova-Schmitz, Starr, et al. 1984 (DNA Protein crosslink)	Rats/Fischer 344/M	0.3, 2, 6, 10, 15 ppm	Nasal tissue Respiratory tissue, Bone Marrow	High	Covalent macromolecular binding was determined using a dual-isotope method ([^3H]CH2O and [^14]CH2O). Macromolecular binding of radiolabeled ^14C-formaldehyde was reported to occur in the respiratory mucosa following inhalation concentrations of 2 ppm and greater. However, no evidence of covalent binding in the olfactory mucosa or bone marrow was reported.
Edrissi et al. 2013 (Protein adduct)	Rat/Fischer 344/M	0, 0.7, 2, 5.8, 9.1 ppm	Nasal tissue	High	Exogenous protein adduct formation was found in the nasal tissue in a concentration-dependent manner following exposure of rats to radiolabeled ^13C^2H2-formaldehyde. However, none detected in the bone marrow.
Heck et al. 1989 (DNA Protein crosslink)	Rat/Fischer 344/M NHP/Macaca mulatta/M	0.3, 0.7, 2, 6, 10 ppm 0.7, 2, 6 ppm	Nasal tissue Nasal tissue, Respiratory tissue, Bone Marrow	High	DNA protein cross links were reported to occur in the nasal tissue of rats following inhalation exposure to radiolabeled ^14C-formaldehyde at concentrations 0.3 ppm and greater. In the monkey, DNA protein cross links were reported to occur in the nasal tissue following exposure to concentrations of 0.7 ppm and greater, but cross links were not detected in the sinus, proximal lung and bone marrow following inhalation exposure to all study concentrations.
Kleinnijenhuis et al. 2013 (Formaldehyde blood concentrations)	Rat/Sprague–Dawley/M	0, 10 ppm	Blood	High	Exogenous labeled ^13C-formaldehyde was not detectable in the blood of rats either during or up to 30 minutes following a 6 hour inhalation exposure. In addition, it was determined that exposure to ^13C-formaldehyde at 10 ppm did not result in an increase in the total formaldehyde concentrations in the blood.
Lai et al. 2016 (DNA Protein crosslink)	NHP/Cynomolgus Macaques/NS Rats/Fisher 344/M	0, 6 ppm (NHP) 0, 15 ppm (Rats)	Nasal tissue, Blood (PBMC), Bone Marrow, Liver	High	Exogenous isotope-labeled ^13CD2-formaldehyde crosslinks were only detected in the nose of nonhuman primates and not detected in the bone marrow, liver or peripheral blood mononuclear cells (PBMC) following inhalation exposure to 6 ppm of formaldehyde. Exogenous isotope-labeled ^13CD2-formaldehyde crosslinks were only detected in the nasal tissue of rats and not the bone marrow or PBMC following inhalation exposure to 15 ppm of formaldehyde for various durations (1, 2 or 4 days).
Leng et al. 2019 (DNA Adduct and DNA Protein Crosslink)	Rats/Fischer 344/M	0, 0.001, 0.03, 0.3 ppm (1, 30, 300 ppb)	Nasal tissue, Bone Marrow, Blood (PBMC), Trachea, Liver Hippocampus, Olfactory bulb, Cerebellum, Lung	High	Exogenous isotope-labeled ^13CD2-formaldehyde adducts or protein crosslinks were not detected any tissue tested following exposure to any test concentration of formaldehyde in rats.
Lu et al. 2011 (DNA Adduct)	Rats/Fischer 344/M	0, 0.7, 2, 5.8, 9.1, 15.2 ppm	Nasal tissue, Bone Marrow	High	Exogenous isotope-labeled ^13CD2-formaldehyde adducts were detected in the nasal tissue of rats exposed to all concentrations of formaldehyde but were not detected in the bone marrow of rats exposed up to 15.2 ppm of formaldehyde.
Lu et al. 2010 (DNA Adduct)	Rats/Fisher 344/M	0, 10 ppm	Nasal tissue, Lung, Liver, Spleen, Bone Marrow, Thymus, Blood	High	Exogenous isotope-labeled ^13CD2-formaldehyde adducts were detected in the nasal tissue of rats exposed to 10 ppm of formaldehyde but were not detected systemically (i.e. in the lung, liver, spleen, bone marrow, thymus or blood).
Moeller et al. 2011 (DNA Adduct)	NHP/Cynomolgus Macaques /NS	0, 2, 6 ppm	Nasal tissue, Bone Marrow	High	Exogenous isotope-labeled ^13CD2-formaldehyde adducts were detected in the nasal tissue with a dose-dependent increase. However, no exogenous formaldehyde adducts were detected in the bone marrow follow exposure to either concentration of formaldehyde.
Speit et al. 2009 (DNA Protein crosslink)	Rats/Fischer 344/M	0, 0.5, 1, 2, 6, 10, 15 ppm	Blood	High	Formaldehyde does not induce DNA protein cross link formation in the peripheral blood of rats following inhalation exposure to formaldehyde based on the lack of DNA migration in the comet assay, as measured after blood from exposed animals following exposure to all concentrations of formaldehyde tested.
Ye et al. 2013 (DNA Protein crosslink)	Mice/BALBc/M	0, 0.4, 0.8, 2.4 ppm (0, 0.5, 1, 3 mg/m^3)	Bone Marrow, Blood, Lung, Liver, Spleen, Testes	High*	DNA protein crosslink formation was reported to occur in the bone marrow, spleen and testes of mice using an assay of protein-linked DNA that is sequentially precipitated from cell lysates following inhalation concentrations of 1 mg/m^3 and greater and in the blood following exposure to 3 mg/m^3 of formaldehyde generated from a formalin solution. The use of formalin for formaldehyde generations renders the study not relevant to the problem formulation questions. Crosslink formation was also reported to occur in the liver following inhalation exposure to all test concentrations of formaldehyde, but no crosslinks were reported to occur in the lung tissue.
Yu R et al. 2015 (DNA adduct)	Rats/Fischer 344/M NHP/Cynomolgus Macaques/NS	0, 2 ppm 0, 2, 6 ppm	Nasal tissue, White Blood Cells, Spleen, Thymus, Lymph nodes, Trachea, Lung, Kidney, Liver, Brain, Bone Marrow	High	Exogenous isotope-labeled ^13CD2-formaldehyde DNA adducts were detected in the nasal tissue or rats following 2 ppm exposure and were not detected in the thymus, lymph nodes, trachea, lung, spleen, kidney, liver, brain or white blood cells. Exogenous formaldehyde adducts were detected in one sample of rat bone marrow tissue analyzed (thought by the study authors to be a methodological error) but was not detected in any other samples from the bone marrow of exposed rats. Exogenous formaldehyde adducts were detected in the nasal tissues of monkeys following exposure to 2 or 6 ppm, but exogenous formaldehyde adducts were not detected in the bone marrow, white blood cells or trachea of monkeys following exposure to either concentration of formaldehyde.

*While scoring high based on systematic review methods, this study was considered not relevant to the problem formulation based on the use of methodologies that did not differentiate between exogenous and endogenous formaldehyde effects and/or the potential of impurities with the test substance due to the use of formalin (i.e. a mixture of water, methanol, and formaldehyde) as the test article.

Table 10. Integration of evidence for postulated MOA 1 – initiation of leukemia by direct damage to hematopoietic stem cells in the bone marrow.

Modified Bradford Hill consideration	Supporting evidence	Potentially inconsistent evidence
Dose-response Temporal concordance	Available evidence provides little, if any, support of a dose-response relationship or temporal concordance for any of the key events in the proposed MOA.	Multiple high-quality inhalation studies in multiple species and across multiple doses that were directly relevant to the problem formulation provide evidence indicating macromolecular binding and genotoxicity in the bone marrow do not occur in animals or humans.
Consistency, specificity	Available evidence provides little, if any evidence of consistency or specificity across studies to support the postulated MOA.	High degree of consistency across studies to support the lack of evidence of macromolecular binding or genotoxicity occurring in the bone marrow after inhalation exposure.
Biological plausibility	Available evidence provides little, if any, evidence for biological plausibility at exposure concentrations that do not overwhelm compensatory mechanisms.	High quality and directly relevant studies in multiple species demonstrate that inhaled formaldehyde does not reach the blood or bone marrow in, measurable quantities and, thus, there is no molecular initiating event to start a leukemogenic process.

Table 11. Formaldehyde-specific dose-response and temporal concordance data for leukemia.

Dose (ppm)	KE: formaldehyde reaches systemic circulation*	KE: macromolecular binding to bone marrow or blood	KE: in vivo genotoxicity^† in HSCs/HPCs or primitive pluripotent cells	KE: damaged stem cell released into circulation	KE: incorporation of initiated cell back into bone marrow
0.2	No data	No data	−^&
0.3	−	No data	−^&	No data	No data
0.4	No data	−^#−^&	±^#	No data	No data
			−^&
0.5	−	−^&	−^#−^&	No data	No data
0.6	No data	No data	−^&	No data	No data
0.7	−	−^#	−^&−^$	No data	No data
0.8	No data	+^#	+^#	No data	No data
		−^&	−^&
1	−	−^&	−^&	No data	No data
2	− −	−^# −^&	−^&−^$	No data	No data
2.4	No data	+^# +^&	+^# +^&	No data	No data
3	No data	No data	−^#	No data	No data
5	No data	No data	−^&	No data	No data
6	− −	−^# −^&	−^&−^$	No data	No data
9	−	−^#	No data	No data	No data
10	− −	−^# −^&	−^&−^$	No data	No data
13.5	No data	No data	−^&	No data	No data
14	No data	−^&	No data	No data	No data
15	− −	−^# −^&	−^# −^&−^$	No data	No data

+: effect observed; −: effect not observed experimentally; ±: effect variably observed.

*Requires analytical/biochemical evidence of delivery, not simply an effect reported systemically. Exogenous formaldehyde was reported to not be present in multiple tissues including the lung, liver, bone marrow, blood, spleen, thymus

^†Genotoxicity for supporting evidence is defined by the study authors to be DNA protein crosslinks formation or biomarkers of oxidative stress (i.e. reactive oxygen species (ROS), malondialdehyde (MDA), measured glutathione (GSH), and cytochrome P450 1A1 and glutathione s-transferase theta 1 expression). ^#bone marrow data. ^&blood data. ^$nasal mucosa

Gray citations – indicate data that provide evidence to support postulated MOA.

Blue citations – indicate data that do not provide evidence to support the postulated MOA.

Yellow citations – indicated data that provide both evidence to support and evidence that does not support the postulated MOA.

Table 12. Integration of Evidence for Each of the Key Events Associated with the Postulated MOAs.

Reference	KE: formaldehyde reaches systemic circulation	KE: macromolecular binding to bone marrow or blood	KE: in vivo genotoxicity* in HSCs/HPCs or primitive pluripotent cells	KE: Damaged stem cell released into circulation	KE: Incorporation of initiated cell back into bone marrow
Evidence to support postulated MOAs	Fox et al. 1985 Walker et al. 1964	Ye et al. 2013 (1.6)^#	Wei et al. 2017 (1.6) Ye et al. 2013 (1.6) Zhang et al. 2013 (1.5) Murrell et al. 2005	Lefrancais et al. 2017	National Institutes of Health 2001
			Murrell et al. 2005
Contradictory evidence of postulated MOAs	Edrissi et al. 2013 (1.6) Kleinnijenhuis et al. 2013 (1.2) Lai et al. 2016 (1.2) Lu et al. 2010 (1.6) Lu et al. 2011 (1.3) Moeller et al. 2011 (1.4) Speit et al. 2009 (1.6) Yu R et al. 2015 (1.4) Leng et al. 2019 (1.4)	Casanova and Heck 1987 (1.5) Casanova-Schmitz, Starr, et al. 1984 (1.5) Edrissi et al. 2013 (1.6) Heck et al. 1989 (1.6) Lai et al. 2016 (1.2) Lu et al. 2010 (1.6) Lu et al. 2011 (1.3) Moeller et al. 2011 (1.4) Speit et al. 2009 (1.6) Yu R et al. 2015 (1.4)	Dallas et al. 1992 (1.6) Kligerman et al 1984 (1.6) Speit et al. 2009 (1.6) Zeller, Neuss, et al. 2011 (1.1)^†Meng et al. (2010) (1.1)	No Data	Abkowitz et al. 2003 McKinney-Freeman and Goodell 2004

*Genotoxicity for supporting evidence is defined by the study authors to be DNA protein crosslinks formation or biomarkers of oxidative stress (i.e. reactive oxygen species (ROS), malondialdehyde (MDA), measured glutathione (GSH), and cytochrome P450 1A1 and glutathione s-transferase theta 1 expression).

^#Values in parentheses are overall study quality scores:

Study quality overall score:

 High: ≥ 1 and <1.7

 Medium: ≥1.7 and <2.3

 Low: ≥2.3 and ≤3

^†Because Zeller, Neuss, et al. (2011) was a controlled human exposure study it was considered directly relevant to the problem formulation.

Dark gray citations – indicate studies that provide indirect relevance to the problem formulation questions.

Blue citations – indicate studies that provide direct relevance to the problem formulation questions, direct evidence to the problem formulation questions because they were conducted in humans.

Light gray citations – studies that have been used to support the postulated MOAs but do not provide any formaldehyde specific data or were conducted in a compromised animal model. These studies were not scored for study quality or relevance.

Table 13. Comparative weight of evidence for postulated MOA 2 – toxicity to circulating blood stem cells and progenitors.

Modified Bradford Hill consideration	Supporting evidence	Potentially inconsistent evidence
Dose-response Temporal concordance	Available evidence provides little, if any, support of a dose-response relationship for any of the key events in the proposed MOA.	Multiple high quality and directly relevant studies from multiple species and across multiple doses, provide evidence indicating macromolecular binding and genotoxicity in the blood do not occur in animals, including humans (i.e. no dose response or temporal concordance demonstrated.)
Consistency, specificity	Available evidence provides little, if any, consistency across studies to support the postulated MOA.	High degree of consistency across studies to demonstrate no macromolecular binding, genotoxicity occurring in the blood.
Biological plausibility	Available evidence provides little, if any, supporting evidence for biological plausibility.	High quality, directly relevant studies in multiple species demonstrate that formaldehyde does not reach the systemic circulation in measurable quantities and, thus, there is no molecular initiating event to start a leukemogenic process. Also, high quality, directly relevant studies indicate there is no evidence that (1) formaldehyde induces genetic damage to HSCs or HPCs in the circulation, or (2) genetically damaged HSCs and HPCs resulting from formaldehyde exposure could reenter the bone marrow.

Table 14. Results from studies measuring endogenous and exogenous DNA-adducts and DNA–protein crosslinks in multiple species.

Reference	Species	Tissue	Conc.	Exposure period*	Adduct(s) measured	Endogenous adducts	Exogenous adducts
Lai et al. 2016^†	Monkey	Bone marrow	0 ppm	2 days	dG-Me-Cys (adducts/10^8 dG)	2.30 ± 0.30	ND
			6 ppm		dG-Me-Cys (adducts/10^8 dG)	1.40 ± 0.46	ND
		Liver	0 ppm		dG-Me-Cys (adducts/10^8 dG)	15.46 ± 1.98	ND
			6 ppm		dG-Me-Cys (adducts/10^8 dG)	11.80 ± 2.21	ND
		Nasal	0 ppm		dG-Me-Cys (adducts/10^8 dG)	3.59 ± 1.01	ND
			6 ppm		dG-Me-Cys (adducts/10^8 dG)	3.76 ± 1.50	1.36 ± 0.20
		PBMC	0 ppm		dG-Me-Cys (adducts/10^8 dG)	1.34 ± 0.25	ND
			6 ppm		dG-Me-Cys (adducts/10^8 dG)	1.57 ± 0.58	ND
	Rat	Bone marrow	0 ppm	4 days	dG-Me-Cys (adducts/10^8 dG)	1.64 ± 0.49	ND
			15 ppm	1 days	dG-Me-Cys (adducts/10^8 dG)	1.80 ± 0.47	ND
				2 days	dG-Me-Cys (adducts/10^8 dG)	1.84 ± 0.61	ND
				4 days	dG-Me-Cys (adducts/10^8 dG)	1.58 ± 0.38	ND
		Nasal	0 ppm	4 days	dG-Me-Cys (adducts/10^8 dG)	6.50 ± 0.30	ND
			15 ppm	1 days	dG-Me-Cys (adducts/10^8 dG)	4.42 ± 1.10	5.52 ± 0.80
				2 days	dG-Me-Cys (adducts/10^8 dG)	4.28 ± 2.34	4.69 ± 1.76
				4 days	dG-Me-Cys (adducts/10^8 dG)	3.67 ± 0.80	18.18 ± 7.23
			2 ppm	28 days	dG-Me-Cys (adducts/10^8 dG)	4.51 ± 1.48	2.46 ± 0.44
				28 days + 168 h post exposure	dG-Me-Cys (adducts/10^8 dG)	3.51 ± 0.16	2.14 ± 1.02
				28 days + 24 h post exposure	dG-Me-Cys (adducts/10^8 dG)	3.78 ± 0.69	2.12 ± 1.00
			2 ppm	7 days	dG-Me-Cys (adducts/10^8 dG)	4.78 ± 0.64	0.96 ± 0.17
Lai et al. 2016^†	Rat	PBMC	0 ppm	4 days	dG-Me-Cys (adducts/10^8 dG)	4.98 ± 0.61	ND
			15 ppm	1 days	dG-Me-Cys (adducts/10^8 dG)	3.26 ± 0.73	ND
				2 days	dG-Me-Cys (adducts/10^8 dG)	3.00 ± 0.98	ND
				4 days	dG-Me-Cys (adducts/10^8 dG)	7.19 ± 1.73	ND
Leng et al. 2019^#	Rat	Bone marrow	0 ppb	28 days	N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	4.83 ± 1.54/ 2.19 ± 0.46	ND//ND
			1 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	4.32 ± 1.21/ 2.28 ± 0.55	ND//ND
			30 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	5.03 ± 1.71/ 1.98 ± 0.42	ND//ND
			300 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	4.42 ± 0.69/ 2.45 ± 0.48	ND//ND
		Cerebellum	0 ppb	28 days	N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.45 ± 0.76/ 2.71 ± 0.87	ND//ND
			1 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.62 ± 0.67/ 2.37 ± 0.68	ND//ND
			30 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.46 ± 0.43/ 2.39 ± 1.60	ND//ND
			300 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.35 ± 0.57/ 2.33 ± 0.73	ND//ND
		Hippocampus	0 ppb	28 days	N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.35 ± 0.56/ 1.81 ± 0.46	ND//ND
			1 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.62 ± 0.74/ 1.87 ± 0.41	ND//ND
			30 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.52 ± 0.82/ 1.63 ± 0.51	ND//ND
			300 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.86 ± 0.76/ 1.94 ± 0.39	ND//ND
Leng et al. 2019^#	Rat	Liver	0 ppb	28 days	N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.48 ± 0.21/ 7.27 ± 1.66	ND//ND
			1 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.57 ± 0.31/ 8.03 ± 1.46	ND//ND
			30 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.44 ± 0.34/ 7.93 ± 1.58	ND//ND
			300 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.60 ± 0.76/ 7.13 ± 1.58	ND//ND
		Lung	0 ppb	28 days	N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	5.25 ± 3.23/ 4.07 ± 1.11	ND//ND
			1 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	3.72 ± 2.20/ 3.99 ± 0.61	ND//ND
			30 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	4.79 ± 3.22/ 3.34 ± 0.67	ND//ND
			300 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	5.06 ± 2.51/ 3.48 ± 0.65	ND//ND
		Nasal mucosa	0 ppb	28 days	N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	3.23 ± 0.85/ 2.66 ± 0.54	ND//ND
			1 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	3.59 ± 0.90/ 2.77 ± 0.61	ND//ND
			30 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	3.27 ± 0.76/ 3.01 ± 0.85	ND//ND
			300 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	3.48 ± 0.83/ 2.85 ± 0.74	ND//ND
		Olfactory bulbs	0 ppb	28 days	N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.51 ± 0.62/ 1.69 ± 0.37	ND//ND
			1 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.74 ± 1.05/ 2.55 ± 0.40	ND//ND
			30 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.84 ± 0.45/ 1.89 ± 0.34	ND//ND
			300 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.59 ± 0.38/ 2.04 ± 0.42	ND//ND
Leng et al. 2019^#	Rat	PBMC	0 ppb	28 days	N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.64 ± 1.03/ 1.96 ± 0.66	ND//ND
			1 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.72 ± 0.73/ 2.08 ± 0.56	ND//ND
			30 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.80 ± 1.11/ 1.88 ± 0.64	ND//ND
			300 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	2.94 ± 1.15/ 1.93 ± 0.85	ND//ND
		Trachea	0 ppb	28 days	N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	3.14 ± 0.61/ 1.52 ± 0.70	ND//ND
			1 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	3.23 ± 1.02/ 2.30 ± 1.03	ND//ND
			30 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	3.34 ± 0.75/ 2.41 ± 0.83	ND//ND
			300 ppb		N2-HOMe-dG (adducts/10^7 dG)/ dG-Me-Cys (adducts/10^8 dG)	3.23 ± 0.47/ 1.99 ± 0.57	ND//ND
Lu et al. 2011^&	Rat	Nasal	0.7 ppm	6 hours	N2-HOMe-dG (adducts/10^7 dG)	3.62 ± 1.33	0.039 ± 0.019
			15.2 ppm		N2-HOMe-dG (adducts/10^7 dG)	4.24 ± 0.92	11.15 ± 3.01
			2.0 ppm		N2-HOMe-dG (adducts/10^7 dG)	6.09 ± 3.03	0.19 ± 0.08
			5.8 ppm		N2-HOMe-dG (adducts/10^7 dG)	5.51 ± 1.06	1.04 ± 0.24
			9.1 ppm		N2-HOMe-dG (adducts/10^7 dG)	3.41 ± 0.46	2.03 ± 0.43
Moeller et al. 2011^&	Monkey	Bone marrow	1.9 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	17.5 ± 2.6	ND
			6.1 ppm		N2-HOMe-dG (adducts/10^7 dG)	12.4 ± 3.6	ND
		Maxilloturbinate	1.9 ppm		N2-HOMe-dG (adducts/10^7 dG)	2.50 ± 0.40*	0.26 ± 0.04
			6.1 ppm		N2-HOMe-dG (adducts/10^7 dG)	2.05 ± 0.54	0.41 ± 0.05
Yu R et al. 2015^@	Monkey	Nasal anterior maxillary	0 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	4.21 ± 0.53	ND
			6 ppm		N2-HOMe-dG (adducts/10^7 dG)	3.80 ± 0.91	0.34 ± 0.12
		Nasal dorsal mucosa	0 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	3.81 ± 1.19	ND
			6 ppm		N2-HOMe-dG (adducts/10^7 dG)	3.62 ± 1.28	0.40 ± 0.07
		Nasal Maxilloturbinate	1.9 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	2.50 ± 0.40	0.26 ± 0.04
			6.1 ppm		N2-HOMe-dG (adducts/10^7 dG)	2.05 ± 0.54	0.41 ± 0.05
		Nasal nasopharynx	0 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	3.48 ± 0.53	ND
			6 ppm		N2-HOMe-dG (adducts/10^7 dG)	3.62 ± 1.34	0.33 ± 0.10
		Nasal posterior maxillary	0 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	3.95 ± 0.74	ND
			6 ppm		N2-HOMe-dG (adducts/10^7 dG)	3.46 ± 1.05	0.36 ± 0.16
		Nasal septum	0 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	3.75 ± 0.32	ND
			6 ppm		N2-HOMe-dG (adducts/10^7 dG)	3.56 ± 0.69	0.39 ± 0.15
		Saline extrusion bone marrow	0 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	5.65 ± 2.12	ND
			6 ppm		N2-HOMe-dG (adducts/10^7 dG)	4.41 ± 1.00	ND
		Scraped bone marrow	0 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	10.18 ± 1.35	ND
			1.9 ppm		N2-HOMe-dG (adducts/10^7 dG)	17.50 ± 2.60	ND
		Scraped bone marrow	6 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	11.00 ± 2.01	ND
			6.1 ppm		N2-HOMe-dG (adducts/10^7 dG)	12.40 ± 3.60	ND
		Trachea carina	0 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	2.69 ± 0.95	ND
			6 ppm		N2-HOMe-dG (adducts/10^7 dG)	2.33 ± 1.12	ND
		Trachea proximal	0 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	2.35 ± 1.05	ND
			6 ppm		N2-HOMe-dG (adducts/10^7 dG)	2.50 ± 1.10	ND
		White blood cells	0 ppm	2 days	N2-HOMe-dG (adducts/10^7 dG)	3.64 ± 1.09	ND
			6 ppm		N2-HOMe-dG (adducts/10^7 dG)	3.79 ± 1.19	ND
Yu R et al. 2015^@	Rat	Bone marrow	0 ppm	28 days	N2-HOMe-dG (adducts/10^7 dG)	3.58 ± 0.99	ND
			2 ppm	14 days	N2-HOMe-dG (adducts/10^7 dG)	2.72 ± 1.36	ND
				21 days	N2-HOMe-dG (adducts/10^7 dG)	2.44 ± 0.96	ND
				28 days	N2-HOMe-dG (adducts/10^7 dG)	3.43 ± 2.20	0.34^$
				28 days + 168h post exposure	N2-HOMe-dG (adducts/10^7 dG)	2.78 ± 1.94	ND
				28 days + 24 h post exposure	N2-HOMe-dG (adducts/10^7 dG)	4.67 ± 1.84	ND
				28 days + 6h post exposure	N2-HOMe-dG (adducts/10^7 dG)	2.41 ± 1.14	ND
				28 days + 72 h post exposure	N2-HOMe-dG (adducts/10^7 dG)	5.55 ± 0.76	ND
				7 days	N2-HOMe-dG (adducts/10^7 dG)	3.37 ± 1.56	ND
		Brain	0 ppm	28 days	N2-HOMe-dG (adducts/10^7 dG)	2.13 ± 0.17	ND
			2 ppm		N2-HOMe-dG (adducts/10^7 dG)	2.35 ± 1.00	ND
		Kidney	0 ppm	28 days	N2-HOMe-dG (adducts/10^7 dG)	2.17 ± 0.60	ND
			2 ppm		N2-HOMe-dG (adducts/10^7 dG)	1.99 ± 0.09	ND
		Liver	0 ppm	28 days	N2-HOMe-dG (adducts/10^7 dG)	1.97 ± 0.38	ND
			2 ppm		N2-HOMe-dG (adducts/10^7 dG)	1.80 ± 0.02	ND
		Lung	0 ppm	28 days	N2-HOMe-dG (adducts/10^7 dG)	2.29 ± 0.24	ND
			2 ppm		N2-HOMe-dG (adducts/10^7 dG)	2.13 ± 0.26	ND
		Lymph nodes	0 ppm	28 days	N2-HOMe-dG (adducts/10^7 dG)	2.99 ± 0.85	ND
			2 ppm		N2-HOMe-dG (adducts/10^7 dG)	2.80 ± 1.38	ND
Yu R et al. 2015^@	Rat	Nasal	0 ppm	28 days	N2-HOMe-dG (adducts/10^7 dG)	2.84 ± 0.54	ND
			2 ppm	14 days	N2-HOMe-dG (adducts/10^7 dG)	3.09 ± 0.98	0.84 ± 0.17
				21 days	N2-HOMe-dG (adducts/10^7 dG)	3.34 ± 1.06	0.95 ± 0.11
				28 days	N2-HOMe-dG (adducts/10^7 dG)	2.82 ± 0.76	1.05 ± 0.16
				28 days + 168h post exposure	N2-HOMe-dG (adducts/10^7 dG)	2.78 ± 0.48	0.67 ± 0.20
				28 days + 24 h post exposure	N2-HOMe-dG (adducts/10^7 dG)	2.98 ± 0.70	0.80 ± 0.46
				28 days + 6h post exposure	N2-HOMe-dG (adducts/10^7 dG)	2.80 ± 0.58	0.83 ± 0.33
				28 days + 72 h post exposure	N2-HOMe-dG (adducts/10^7 dG)	2.99 ± 0.63	0.63 ± 0.12
				7 days	N2-HOMe-dG (adducts/10^7 dG)	2.51 ± 0.63	0.35 ± 0.17
		Spleen	0 ppm	28 days	N2-HOMe-dG (adducts/10^7 dG)	2.18 ± 0.19	ND
			2 ppm		N2-HOMe-dG (adducts/10^7 dG)	1.83 ± 0.25	ND
		TBLN	0 ppm	28 days	N2-HOMe-dG (adducts/10^7 dG)	3.46 ± 1.24	ND
			2 ppm		N2-HOMe-dG (adducts/10^7 dG)	3.01 ± 0.71	ND
		Thymus	0 ppm	28 days	N2-HOMe-dG (adducts/10^7 dG)	0.78 ± 0.04	ND
			2 ppm		N2-HOMe-dG (adducts/10^7 dG)	0.63 ± 0.06	ND
		Trachea	0 ppm	28 days	N2-HOMe-dG (adducts/10^7 dG)	3.18 ± 0.72	ND
			2 ppm		N2-HOMe-dG (adducts/10^7 dG)	2.63 ± 0.92	ND
		White Blood Cells	0 ppm	28 days	N2-HOMe-dG (adducts/10^7 dG)	2.76 ± 0.66	ND
			2 ppm	14 days	N2-HOMe-dG (adducts/10^7 dG)	2.26 ± 0.46	ND
				21 days	N2-HOMe-dG (adducts/10^7 dG)	2.40 ± 0.47	ND
				28 days	N2-HOMe-dG (adducts/10^7 dG)	2.49 ± 0.50	ND
				28 days + 168h post exposure	N2-HOMe-dG (adducts/10^7 dG)	2.61 ± 1.22	ND
Yu R et al. 2015^@	Rat	White Blood Cells	2 ppm	28 days + 24 h post exposure	N2-HOMe-dG (adducts/10^7 dG)	2.57 ± 0.58	ND
				28 days + 6h post exposure	N2-HOMe-dG (adducts/10^7 dG)	2.97 ± 0.58	ND
				28 days + 72 h post exposure	N2-HOMe-dG (adducts/10^7 dG)	1.75 ± 0.26	ND
				7 days	N2-HOMe-dG (adducts/10^7 dG)	2.62 ± 1.12	ND

*Exposure occurred for 6 h per day over the duration of the exposure period. ^†Limit of detection was 37.5 amol on the column. ^#Limit of detection was 0.5 amol on the column for N2-HOMe-dG; 5 amol on the column for dG-Me-Cys. ^&Limit of detection was 20 amol on the column. ^@Limit of detection was 10 and 0.5 amol on Thermo TSQ Quantum and AB SCIEX Triple Quad 6500, respectively.

^$Exogenous adducts were detected in one bone marrow sample across numerous studies and the study authors have indicated it is likely an error.

Table 15. Comparative weight of evidence for postulated MOA 3 and 4 – targeting pluripotent nasal cells or HSCs/HSPs in lung tissue.

Modified Bradford Hill consideration	Supporting evidence	Potentially inconsistent evidence
Dose-response temporal concordance	No chemical-specific data available to provide support for dose-response or temporal concordance	Dosimetry demonstrates the mass of inhaled exogenous formaldehyde is insignificant compared the body burden of endogenously produced formaldehyde. Temporal concordance of leukemia development has not been identified in animal or human studies.
Consistency, specificity	No chemical specific data available that provides consistent support for the postulated key events.	Dosimetry data demonstrates lack of exposure to target cell populations, and are specific to formaldehyde and consistent across multiple studies
Biological plausibility	Plausible, but no chemical-specific data to support and not generally accepted as a relevant AOP for leukemia development	Pluripotent stem or HSPs/HSC cell data are limited to animal models with compromised systems and are not formaldehyde-specific.

Table 16. Comparison of formaldehyde regulatory standards, exogenous formaldehyde concentrations in food and drink, and endogenous production of formaldehyde in humans.

Exposure type	Exposure assumptions		Equivalent daily exposure (mg/day)	Reference/source
	Source	Value
Regulatory inhalation standards
Indoor air	WHO Indoor Air Quality Guideline	0.1 mg/m^3	2*	World Health Organization 2010; Nielsen et al. 2017
Workplace Air	OSHA PEL Standard	0.9225 mg/m^3	9^†	Occupational Safety and Health Administration 2020
Exogenous sources of formaldehyde from common food
Fruits including apples,  carrots, watermelon,  apricot, tomatoes  and bananas)	Metabolic demethylation of pectin	6–20 mg/kg/day	420–1400^#	European Food Safety Authority 2014
Total from all food sources	Ingestion of 1 kg of food per day	1.4 mg/kg/day	100^#	European Food Safety Authority 2014
Endogenous production
Diet soft drink (12 oz)	Aspartame Demethylation	4 mg/kg/day	280^#	European Food Safety Authority 2014
Total endogenous  production	All metabolic sources	70 kg bw	61,460–91,700	European Food Safety Authority 2014

*Based on an adult daily inhalation rate of 20 m³/day and a body weight of 70 kg. ^†Based on an occupational inhalation rate of 10 m³/day and a body weight of 70 kg. ^#Assuming an average adult body weight of 70 kg.

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