An updated mode of action and human relevance framework evaluation for Formaldehyde-Related nasal tumors

Figures & data

Table 1. Nasal tumor incidence in formaldehyde inhalation studies.

Strain	Sex	n/group	Duration	0^a	0.1	0.3	0.7	1	2	5.6/6	10	12.4	14/15	30	41	82	163	Study
RATS
Wistar	M	10	1 year	0/10	0/10	–	–	0/10	–	–	0/10	–	–	–	–	–	–	Appelman et al. (1988)
S-D	F	16	2 year	0/15	–	–	–	–	–	–	–	1/16	–	–	–	–	–	Holmstrom et al. (1989)
F344	M	32	2 year	0/32	–	0/32	–	–	0/32	–	–	–	13/32	–	–	–	–	Kamata et al. (1997)
S-D	M	100	Life	0/99	–	–	–	–	–	–	–	–	38/100	–	–	–	–	Sellakumar et al. (1985)
F344	MF	120	2 year	0/232	–	–	–	–	0/236	2/235	–	–	103/232	–	–	–	–	Swenberg et al. (1980) Pavkov et al. (1982) Kerns et al. (1983)
F344	MF	90-150	2 year	0/90	–	–	0/90	–	0/96	1/90	20/90	–	69/147	–	–	–	–	Monticello et al. (1996)
Wistar	M	30	2 year	0/30	1/30	–	–	1/30	–	–	1/30	–	–	–	–	–	–	Woutersen et al. (1989)
MICE
C3H	?	60	35 weeks		–	–	–	–	–	–	–	–	–	–	0%	0%	0%	Horton et al. (1963)
B6C3F1	MF	120	2 year	0/240	–	–	–	–	0/240	0/240	–	–	2/240	–	–	–	–	Swenberg/Kerns
HAMSTERS
Syrian	M	88-132	Life	0/132	–	–	–	–	–	–	0/88	–	–	–	–	–	–	Dalbey (1982)
Syrian	M	50	Life	0/50	–	–	–	–	–	–	–	–	–	0/50	–	–	–	Dalbey (1982)

^a Doses are in ppm formaldehyde.

Figure 1. Rat nasal passages, tumor response, and dosimetry. (A) Diagrams of rodent nasal cavity demonstrating prominent bone structures and epithelial lining of the rat nasal cavity (adapted from Alvites et al. 2018). Diagram of the various Levels (I–IV) of the sagittal section of the rat nasal cavity (adapted from Kerns et al. 1983). The lower portion shows coronal sections at LI, LII and LIII, where the white represents air passages (meatuses) including the lateral meatus (L) and medial meatus (MM); the black represents bone (lined by epithelium), with nasal turbinates (N), maxilloturbinates (MT) and septum (S) (adapted from Harkema et al. 2006; reprinted by Permission of SAGE Publications, Inc.). The red circle at LII corresponds to the tumor shown in C, whereas the shaded blue region corresponds to the CFD flux estimates in D. (B) Dose-response for nasal tumors two animal bioassays (data adapted from U.S. EPA (2010)). Green vertical line represents the dose (ppm) at which formaldehyde would increase tumor “extra risk” by 10% (data plotted using BMDS v3.1). (C) H&E stained early squamous cell carcinoma (arrow) arising from the nasoturbinates of a rat exposed to 15 ppm formaldehyde (adapted from Swenberg et al. 1980). The red circle corresponds to the red circle in A. (D) Map of simulated formaldehyde flux along airway walls based on CFD modeling in rat nose (coronal section) at 252 ml/min airflow (reprinted from Kimbell et al. (1993) with permission from Elsevier). M: maximum mass flux at walls.

Figure 2. Mapping of regions of various effects of formaldehyde exposure and tissue collection regions. The percentage of nasal SCC in the rat cancer bioassays are shown on the left and color coded to the levels and anatomical regions (left air passage) shown in the middle diagram. Tissue harvest locations from select studies are listed on the right along with color coding to the levels and anatomical regions (right nasal passage) shown in the middle diagram. The shaded blue trapezoid region corresponds to tissue harvested for select assays listed on the right. Diagram of the sagittal section of the rat nasal cavity and coronal sections are adapted from Kerns et al. (1983) and Harkema et al. (2006), (reprinted by Permission of SAGE Publications, Inc.) respectively. See legend of Figure 1 for acronyms. Note: these color codings are interpretations by the authors based on the reported data and are for illustrative purposes only.

Figure 3. Cell proliferation in rat nasal tissue following subchronic and chronic inhalation exposure to formaldehyde. (A) Unit length labeling index in various regions of the nasal cavity following inhalation exposure to 0.7–15 ppm formaldehyde for 3–18 months (administration of [³H]thymidine by osmotic pumps). Data from Monticello et al. (1996). (B) Unit length labeling index in various regions of the nasal cavity following inhalation exposure to 0.7–15 ppm formaldehyde for 1–42 days (administration of [³H]thymidine by i.p. injection). Data from Monticello et al. (1991). ALM: anterior lateral meatus; PLM: posterior lateral meatus; AMS: anterior mid-septum; PMS: posterior mid-septum; MMX: medial maxilloturbinate.

Figure 4. Diagram of formaldehyde metabolism and select adduct formation. See text for various acronyms. Cofactors such oxidized and reduced nicotinamide adenine dinucleotide (NAD⁺ and NADH) are not shown for simplicity. Formaldehyde is shown crosslinking deoxyguanine (dG) and the cysteine in the tripeptide GSH as an example of relatively stable -N-Me-S- formaldehyde linkages that form between DNA and proteins (DPC). Such DPC have been shown to undergo hydrolysis to HmdG. Examples of mass differences between exogenous (m/z = 285.2) and endogenous (m/z = 282.2) HmdG (in box), which can also undergo hydrolysis back to dG. The mM levels of formaldehyde and GSH are from Andersen et al. (2010).

Table 2. Estimated nasal tissue dose in rats.

Formaldehyde (ppm)	Formaldehyde (mg/m^3)	Intake (m^3/6 h)	Dose (mg/6 h)
0.001	0.001	0.077	9.45E-05
0.03	0.037	0.077	0.00283
0.3	0.37	0.077	0.0283
0.7	0.86	0.077	0.0661
2	2.5	0.077	0.189
6	7.4	0.077	0.567
10	12	0.077	0.945
15	18	0.077	1.42

Bold values indicated inhalation exposure and tissue doses flanking internal dose of 0.22 mg.

Figure 5. Summary of formaldehyde-DNA and formaldehyde-protein adducts following inhalation exposure to formaldehyde. Studies show the progression of exposure technology from unlabeled-formaldehyde to dual heavy isotope [¹³CD₂]-formaldehyde, and progression of analytical technology from liquid scintillation counting (LSC) to HPLC technologies, mass spectrometry technologies and most recently to nano-LC-MS/MS methods following inhalation exposure to formaldehyde. Note: Casanova & Heck (1987) exposed rats after pretreatment with corn oil (+GSH) or the GSH inhibitor phorone (−GSH). Open symbols represent doses where DPC or related adducts were not observed at experimental concentrations. Triangles represent non-human primates (all other data are in rats). Dotted line represents 0.3 ppm.

Figure 6. Measures of endogenous and exogenous DNA adducts. (A) HmdG in nasal tissue of rats exposed to various concentrations of formaldehyde [¹³CD₂]-formaldehyde. Filled black circles indicate HmdG levels after 6 h exposure to 0.7, 2, 6, 10, and 15 ppm [¹³CD₂]-formaldehyde. Red triangles represent exogenous HmdG levels after 28 days of exposure. All other symbols represent HmdG levels after exposure to 2 ppm [¹³CD₂]-formaldehyde for indicated lengths of time. The dotted line indicates the ±1 s.d. range on of endogenous HmdG levels reported in Lu et al. (2010); other data taken from Lu et al. (2011), Yu et al. (2015), and Leng et al. (2019). Exogenous adducts were not detected (ND) at ≤0.3 ppm therefore the adducts levels were set to the limit of detection (LOD). The dotted line indicates the ±1 s.d. range on endogenous adducts. (B) Linear scale plot of the 6 h data (black circles) and 28-day low dose data (red diamonds). The two linear segments in the main plot are the result of segmental linear regression in Prism, which is used here to accentuate the applied concentrations that result in a change in slope (other nonlinear models are not explored here). The inset shows the ratio of exogenous HmdG to endogenous HmdG, with the dashed line indicating unity.

Table 3. In vivo genotoxicity studies on formaldehyde.

Endpoint	Species/sex	Route	Dose	Duration/exposures	Analysis time	Tissue	Results	TSCA Score^a	Reference
MN	SD rats; M	p.o.	200 mg/kg	NS (single?)	16 h, 24 h, 30 h	Stomach, duodenum, ileum, colon	+	1.5	Migliore et al. (1989)
MN	NMR1 mice; M, F	i.p.	10, 20, 30 mg/kg	Single	NS	BM	−	4	Gocke et al. 1981
CA	Q strain mice; M	i.p.	50 mg/kg	Single	9–15 days	Spermatocytes	−	1.9	Fontignie-Houbrechts 1981
MN	CBA mice; M, F	i.p.	6.25, 12.5, 25 mg/kg	2× (0 and 24 h)	16 hr, 40 hr	BM PCE	−	1.7	Natarajan et al. 1983
CA	CBA mice; M, F	i.p.	6.25, 12.5, 25 mg/kg	2× (0 and 24 h)	16 hr, 40 hr	BM & spleen	−	1.7	Natarajan et al. 1983
CA	F344 rats; M, F	inhalation	0.5, 6, 15 ppm	6 h/day × 5 days	1 hr	Blood lymphocytes	−	1.3	Kligerman et al. 1984
SCE	F344 rats; M, F	inhalation	0.5, 6, 15 ppm	6 h/day × 5 days	1 hr	Blood lymphocytes	−	1.3	Kligerman et al. 1984
CA	SD rats; M	inhalation	0.5, 3, 15 ppm	6 h/day × 1 week or 8 week	NS	Lung lavage	+	1.4	Dallas et al. 1992
CA	SD rats; M	inhalation	0.5, 3, 15 ppm	6 h/day × 1 week or 8 week	NS	BM	−	1.4	Dallas et al. 1992
Comet	F344 rats; M	inhalation	0.5, 1, 2, 6, 10, 15 ppm	6 h/day × 5/week × 4 wk	NS	Peripheral blood	−	1.2	Speit et al. 2009
SCE	F344 rats; M	inhalation	0.5, 1, 2, 6, 10, 15 ppm	6 h/day × 5/week × 4 week	NS	Peripheral blood	−	1.2	Speit et al. 2009
MN	F344 rats; M	inhalation	0.5, 1, 2, 6, 10, 15 ppm	6 h/day × 5/week × 4 week	NS	Peripheral blood	−	1.2	Speit et al. 2009
mutation	F344 rats; M	inhalation	0.7, 2, 6, 10, 15 ppm	6 h/day × 5/week × 13 week	NS	Nasal mucosa (Level II)	−	1.3	Meng et al. 2010
Comet	F344 rats; M	inhalation	0.5, 1, 2, 6, 10, 15 ppm	6 h/day × 5/week × 4 week	NS	Broncho-alveolar lavage (BAL)	−	1.3	Neuss et al. 2010
MN	F344 rats; M	inhalation	0.5, 1, 2, 6, 10, 15 ppm	6 h/day × 5/week × 4 wk	NS	Broncho-alveolar lavage (BAL)	−	1.3	Neuss et al. 2010
MN	F344 rats; M	inhalation	0.5, 1, 2, 6, 10, 15 ppm	6 h/day × 5/week × 4 week	NS	Nasal epithelium	−	1.5	Speit, Schutz, et al. 2011
MN	male & female volunteers	inhalation	0.15-0.5 ppm with peaks of 15-min at 1 ppm	4 h/day × 2 week	0, 7, 14, 21 days after exposure	Buccal cells	−	1.0	Speit et al. 2007
MN	male nonsmokers	inhalation	0.3, 0.4, 0.5, 0.7 with peaks up to 0.8 ppm	4 h/day × 5 days	After last exposure	Peripheral blood	−	1.3	Zeller et al. 2011
SCE	male nonsmokers	inhalation	0.3, 0.4, 0.5, 0.7 with peaks up to 0.8 ppm	4 h/day × 5 days	After last exposure	Peripheral blood	−	1.3	Zeller et al. 2011
Comet	male nonsmokers	inhalation	0.3, 0.4, 0.5, 0.7 with peaks up to 0.8 ppm	4 h/day × 5 days	After last exposure	Peripheral blood	−	1.3	Zeller et al. 2011
MN	male nonsmokers	Inhalation	0.3, 0.4, 0.5, 0.7 with peaks up to 0.8 ppm	4 h/day × 5 days	After last exposure	Nasal epithelial swabs	−	1.3	Zeller et al. 2011
Comet	Unspecified mice^a; M	Inhalation	16, 33, 65 ppm	2 h/day × 15 day	After last exposure	BM	+	4	Yu, Song, et al. (2015)
MN	ICR mice; M	Inhalation	16, 33, 65 ppm	2 h/day × 15 day	After last exposure	BM	+	4	Yu et al. (2014)
MN	ICR mice; M	Inhalation	1, 10 mg/m^3	2 h/day × 20 week	24 h after last exposure	BM	−	4	Liu et al. (2017)
Tumor	C3B6.129F1-Trp53^tm1Brd	Inhalation	7.5, 15 ppm	6 h/day × 5/week × 8 week	∼32 week	Nasal mucosa	−	1.1	Morgan et al. 2017
Tumor	B6.129-Trp53^tm1Brd	Inhalation	7.5, 15 ppm	6 h/day × 5/week × 8 week	∼32 week	Nasal mucosa	−	1.1	Morgan et al. 2017

^aTSCA Overall Quality Sore ranges: High (≥1–1.7), Medium (≥1.7–<2.3), Low (≥2.3–≤3), Unacceptable (4); note: TSCA scores for studies that were also scored in Gentry et al. 2021 resulted in the same overall quality grade, albeit with slightly different numerical values due to independent scoring. Abbreviations: MN: micronucleus; SCE: sister chromatid exchange; BM: bone marrow; PCE: polychromatic erythrocytes; NS: not specified; M: male; F: female.

Figure 7. ADH5 null mice. (A) Mutant frequency in Big Blue^® (WT) mice and ADH5/GSNOR deficient Big Blue^® mice (GSNOR^−/−). Adapted from Leung et al. (2013). (B) Mice deficient in ADH exhibit higher levels of endogenous HmdG in multiple tissues. Adapted from Pontel et al. (2015). Data were extracted from published figures with WebPlotDigitizer 4.3.

Figure 8. MOA for SCC in the rodent nasal cavity. KE1: saturation of formaldehyde metabolism results in increased free formaldehyde which increases adduction to cellular molecules (e.g. protein, DNA). KE2: increased adduction leads to irritation and cytotoxicity (KE2a) and/or DNA damage (KE2b). KE3: squamous metaplasia is an adaptive response that can be reversible if exposure ceases or decreases (e.g. ≤1 ppm for formaldehyde), can persist if the metaplasia protects against continued exposure (e.g. 2–6 ppm for formaldehyde), or can be overwhelmed if higher exposures (e.g. ≥6 ppm for formaldehyde) exceed protection afforded by squamous epithelium. KE4: continued exposure to cytotoxic concentrations (after adaptive metaplasia has occurred) leads to chronic cell proliferation. KE5: increased cell replication and potentially increased DNA damage increase mutations during replication. The blue “X” indicates that data published after McGregor et al. (2006) do not support a direct contribution from formaldehyde-induced DNA lesions (see text).

Table 4. Proposed MOA for formaldehyde-induced nasal tumors.

Key event	Brief description
1. Saturation of formaldehyde metabolism leading to increased free formaldehyde and adduction	Data indicate that there are exposure concentrations that do not result in detectible levels of exogenous formaldehyde (e.g. HmdG) in nasal tissue. Various extracellular barriers (e.g. mucus) could plausibly result in non-detects for biomarkers of exogenous formaldehyde. Transcriptomic responses (biomarkers of effect) indicate no cellular response to 0.7 ppm formaldehyde. Models indicate that formaldehyde saturation begins to occur between 2 and 4 ppm. Transcript changes begin to occur around 2 ppm in naïve non-squamous mucosa at 6 ppm in squamous epithelium. Exogenous HmdG levels do not reach endogenous HmdG levels until ∼6 ppm. At ≥6 ppm, HmdG levels begin to exceed endogenous levels (i.e. increase the total burden >2-fold).
2a. Irritation/Cytotoxicity (See Figure 8)	Elevations in free formaldehyde lead to cytotoxicity and subsequent proliferative effects and squamous metaplasia as evidenced by H&E staining and cell proliferation (e.g. ULLI).
2b. DNA damage (potentially pro-mutagenic) The weight of evidence for direct DNA damage is weak. (See Figure 8)	Elevations in free formaldehyde lead to increased adduction to cellular components, including protein and DNA. DPC represent one form of formaldehyde-related DNA lesion. Generally, DPC are associated with genotoxicity (including mutagenicity); however, data published since 2006 indicate little/no direct evidence of genotoxicity in the nasal cavity. The new data demonstrate that exposure to ≤15 ppm formaldehyde does not induce genotoxicity in rat nasal tissue or neoplasms in p53^+/- mice. ADH5^-/- mice have elevated HmdG levels yet no increases in γ-H2AX, CA, or MF in tissues examined (nasal tissue has not been examined). Exposure to 15 ppm formaldehyde clearly results in HmdG levels above endogenous levels; however, there is little/no evidence of genotoxicity but clear evidence of cytotoxicity.
3. Squamous Metaplasia	Adaptive squamous metaplasia can reverse upon cessation of exposure or persist if irritating exposure continues to occur. Squamous metaplasia creates an epithelium more resistant to chemical and physical irritation.
4. Cytotoxicity/regenerative cell proliferation	After the onset of adaptive metaplasia, continued exposure to high concentrations of formaldehyde (≥6 ppm) result in cytotoxicity and regenerative cell proliferation. This is supported by transcriptomic responses and increased cell proliferation (e.g. ULLI).
5. Mutations (replication error)	A lifetime increase in cell replications can increase the chance for mutations to form, which occur with some probability during each cell division. BBDR models indicate that nasal tumor formation in rats can be fitted without a mutagenic component. In vivo genotoxicity studies in nasal tissue have not detected genotoxic damage (see description for KE2b).
SCC in rodent nasal cavity.	Borrowing from AOP terminology, the SCC is an adverse outcome (AO) as opposed to a key event. These tumors were only observed in rats at ≥6 ppm formaldehyde.

All acronyms are defined in the main text.

Table 5. Summary of metaplasia in nasal cavity of rats in LI-III.

	Formaldehyde (ppm)
Study	0.7	2	6	10	15
Andersen et al. (2008) (5 days)	–	–	I-III	nd	nd
Andersen et al. (2008) (15 days)	–	–	–	nd	nd
Andersen et al. (2010)** (4–13 weeks)	–	I	I, II	I, II	I, II
Kerns et al. (1983) (6–24 months)	nd	I	I, II	nd	I–III
Monticello et al. (1996)* (2 years)	–	–	II	II, III	II, III

nd: dose was not included in the study; –: absent or transient/sporadic.

*Level I not examined/reported.

**Level III not examined/reported.

Table 6. Dose and temporal concordance table.

		Temporal
	Dose (ppm)	Dosimetry to Nasal Cells (min-hours)**	KE1. Saturation of FA metabolism, FA adduction (hours-days)	KE2a. Irritation/cytotoxicity (hours-days)	KE2b. DNA damage (pro mutagen?) (hours-days)	KE3. Squamous Metaplasia (days-weeks)	KE4. Cytotoxicity/regenerative cell proliferation (days-weeks)	KE5. Mutations (replication error) (months-years)	SCC (∼1 year+)
Dose	≤0.03	−
	0.3	+/−	−
	0.7	+	−	−	−	−	−	−	−
	1	+*	−	−*	−	−	−*	−	−*
	2	+	+/−	+/−	−	+	−	−	−
	6	+	+	+	−	+	+	−	+/−
	10	+	+	+	−	+	+	−	+
	15	+	+	+	−	+	+	+/−***	+
	Notes:	Figures 5 & 6			Table 3 Figure 7	Table 5	Figure 3	Table 3 BBDR model	Figure 1B

+: Effect observed; −: effect not observed experimentally; ±, effect variably observed (for tumors incidence was not significant by one-sided FET).

blank: effect not studied at specific dose; shading indicates region where effect is not observed.

*Inferred based on lower dose.

**Times in parentheses indicate approximate onset.

***Although the available data (including BBDR modeling) do not indicate genotoxicity, HmdG adducts at 15 ppm are four-fold above endogenous HmdG levels.

Table 7. Human relevance.

Key event or outcome	Rat	Monkey	Human
Dosimetry of exogenous formaldehyde to nasal epithelial cells	Data indicate there are levels of formaldehyde exposure that do not reach DNA (e.g. no exogenous HmdG). Clear dose-dependent increases in DPC and HmdG >0.3 ppm	Dose-dependent increases in DPC and HmdG are observed, but ultra-low exposures needed to explore threshold have not been conducted in NHP (yes; threshold likely)	No data using sensitive labeled formaldehyde assays (yes; threshold likely)
1. Saturation of formaldehyde metabolism leading to increased free formaldehyde and adduction to cellular components	Models indicate inflections in free formaldehyde between 2 and 6 ppm Exogenous HmdG levels increase linearly until they reach endogenous levels at 6-10 ppm, then increase more steeply up to 15 ppm Transcriptomic changes mostly occur at ≥6 ppm in tissue that has undergone squamous metaplasia	No data (yes; conservation of ADH5 suggests formaldehyde-GSH metabolism also important; increased DPC observed in monkeys, but at lower levels relative to rats)	No data (yes; conservation of ADH5 suggests formaldehyde-GSH metabolism also important; increased DPC observed in monkeys, but at lower levels relative to rats)
2. Irritation/Cytotoxicity	Adaptive histopathological changes observed at ≥2 ppm. Transcriptomic changes mostly occur at ≥6 ppm; changes at 2 ppm are more modest.	No histopathological changes at 0.2 ppm; minimal metaplasia at 1 ppm; 100% incidence at 3 ppm (yes; nonlinearities are evident)	Mixed data from occupational studies. (CFD/BBDR models can estimate air concentrations and/or internal doses similar to cytotoxic doses in monkeys or rats; nonlinearities are likely)
3. Squamous metaplasia	Adaptive histopathological changes observed at ≥2 ppm. Persistent metaplasia occurs at 2 ppm without quantitative increases in cell proliferation. Squamous epithelium is not completely protective at ≥6 ppm.	No histopathological changes at 0.2 ppm; minimal metaplasia at 1 ppm; 100% incidence at 3 ppm (yes; nonlinearities are evident)	Mixed data from occupational studies. (yes; CFD/BBDR models can estimate air concentrations and/or internal doses resulting in squamous metaplasia in monkeys or rats; nonlinearities are likely)
4. Cytotoxicity/Regenerative cell proliferation	After squamous metaplasia has occurred, increased cell proliferation (e.g. ULLI) is variably observed at 6 ppm and significantly increased at ≥10 ppm	LI increased at ≥6 ppm (yes; nonlinearities are evident)	No labeling data (yes; CFD/BBDR models can estimate air concentrations and/or internal doses resulting in regenerative cell proliferation in monkeys or rats; nonlinearities are likely)
5. Mutations (replication error)	No direct evidence of mutagenic or clastogenic effects in exposed rats in short-term assays. No increases in mutation frequency in ADH5/GSNOR null Big Blue mice. No neoplasms in p53^+/− mice. BBDR model can link inhalation exposure to internal doses that increase acquisition of mutations in clonal growth model. The role of DPC could also be modeled if the relationship between DPC and mutation rate were known.	No direct evidence of mutations in NHP (yes, nonlinearities are likely)	Some data from occupational studies; however, no evidence of genotoxicity in controlled human exposure studies. (yes; CFD/BBDR models can estimate the internal dose to humans that is similar to internal doses in rats that increase acquisition of mutations and tmors; nonlinearities are likely)
Nasal tumor formation	SCC increased at ≥6 ppm	No long-term studies (yes, nonlinearities are likely)	Controversial evidence for increased NPC in workers exposed to formaldehyde. (yes; CFD/BBDR models can estimate air concentrations and/or internal doses resulting in similar internal doses to rats that increase tumor formation; nonlinearities are likely)

Note: “Yes” refers to whether the KE is likely to occur in species.

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