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Basic toxicokinetics

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basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference Type:
Formaldehyde: Integrating Dosimetry, Cytotoxicity, and Genomics to Understand Dose-Dependent Transitions for an Endogenous Compound
Andersen, M.E. et al.
Bibliographic source:
Tox. Sci. 118(2), 716–731

Materials and methods

Objective of study:
Principles of method if other than guideline:
Combined studies with different FA exposure levels and exposure duration with toxicokinetic modelling for tissue formaldehyde acetal (FAacetal and glutathione levels and with histopathology and gene expression in nasal epithelium from rats exposed to 0, 0.7, 2, 6, 10 or 15 ppm formaldehyde for 6 hours/day for 1, 4 or 13 weeks.
GLP compliance:
not specified

Test material

Test material form:
Specific details on test material used for the study:
- Name of test material (as cited in study report): Formaldehyde (FA)
- Substance type: Paraformaldehyde (CAS# 30525-89-4) was vaporized
- Analytical purity: 94.5–95.0% pure

Test animals

Fischer 344
Details on test animals and environmental conditions:
- Source: Charles River Breeding Laboratories, Inc. (Wilmington, MA or Kingston, NY).
- Age at study initiation: 6 to 7 weeks
- Housing: Individually
- Acclimation period: 2 weeks

Administration / exposure

Route of administration:
inhalation: vapour
other: Nitrogen/Air
Details on exposure:

Test atmospheres were generated by slowly vaporizing solid paraformaldehyde in stainless steel pans of various sizes in sealed stainless steel canisters in an oven. Nitrogen flowed through the stainless steel canister and carried vaporized FA into the charcoal-HEPA-filtered air supplying each chamber.
Atmosphere concentrations were monitored every 30 min during the 6-h exposure period, using a calibrated infrared (IR) analyzer (Miran 1A, Foxboro, MA). A calibrated IR spectrophotometer was assigned to each of six exposure chambers (including the control chamber). Spectrophotometers were calibrated by injecting known volumes of a bag standard into the closed loop of the IR analyzing cell. The bag standard was made by heating a known mass of paraformaldehyde in a sealed, glass three-neck flask to the point of sublimation and metering a known volume of nitrogen through the flask carrying the FA vapor into a Teflon bag. A chromatropic acid assay (CAA) was used to verify the accuracy of the bag standard. Concentrations analyzed by CAA were used to calibrate each IR spectrophotometer. The limit of detection, based on calibration curves, ranged from 0.066 to 0.074 ppm for the control and low (0.7 ppm) FA concentration chambers.

Duration and frequency of treatment / exposure:
6 h/day for 1, 4, or 13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
0.7 ppm
Dose / conc.:
2 ppm
Dose / conc.:
6 ppm
Dose / conc.:
10 ppm
Dose / conc.:
15 ppm
No. of animals per sex per dose:
- 8 per dose per time period for the first 13-week study (cell proliferation and histopathology) for the second 13-week study (gene expression, cytokines, hematology, and bone marrow evaluation).
- 15 per dose per time point for the second 13-week study (gene expression, cytokines, hematology, and bone marrow evaluation).
Control animals:
yes, concurrent vehicle
Details on study design:
Concentration and exposure duration transitions in FA mode of action (MOA) were examined with pharmacokinetic (PK) modeling for tissue formaldehyde acetal (FAcetal) and glutathione (GSH) and with histopathology and gene expression in nasal epithelium.

A simplified flux-based PK model that described endogenous FAcetal and GSH and to use the model to estimate background FAcetal, increasing tissue FAcetal with increasing concentrations of inhaled FA, and the increases in 14C-DNAprotein cross-links seen in earlier studies was developed within the study.
- Both endogenous and exogenous FA was included by an external input from FA over a range of 0–15 ppm to simulate inhalation and a dose-independent input by production of FAcetal by cellular metabolism.
- Input into each side of the nose was estimated as the product of half the ventilation rate times the inhaled FA concentration.
- The 14C-DNA-protein cross-link data are a surrogate for tissue 14C-FAcetal. This PK description had reversible binding of cellular FAcetal with GSH.
Total GSH (2.8mM) and FA (0.4mM) had been previously measured (Casanova-Schmitz et al., 1984; Heck et al., 1982). These two estimates were key input parameters that could not be altered by the choice of model parameters.
- A published dissociation constant for the formaldehyde thioacetal, 1.5mM was used (Keller et al., 1990)
- The zero-order production rate of FA by catabolism and the maximum rate of oxidation of FA by FDH were fit to simultaneously match steady-state
tissue FA.
- The model for radiolabeled DNA-protein cross-links was structured to describe a tracer input.
- he model for 14C-FA had no metabolic input and used the concentrations of unlabeled GSH and FSG for the reactions involving formation of the thioacetal and metabolism by FDH.
- The 14C-DNAprotein cross-links were described by multiplying the simulated 14C-FAcetal by a fitted constant to match the literature data.
Details on dosing and sampling:
Nasal cavity cell proliferation and histopathology:

Results and discussion

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
It was concluded that dose dependencies, high background levels of FA acetal, and nonlinear FA acetal/glutathione tissue kinetics indicated that FA concentrations below 1 or 2 ppm would not increase the risk of cancer in the nose or any other tissue, or affect FA homeostasis within epithelial cells.

Any other information on results incl. tables

- Patterns of gene expression varied with concentration and duration.

- At 2 ppm, sensitive response genes (SRGs)—associated with cellular stress, thiol transport/reduction, inflammation, and cell proliferation—were upregulated at all exposure durations.

- At 6 ppm and greater, gene expression changes showed enrichment of pathways involved in cell cycle, DNA repair, and apoptosis.

- ERBB, EGFR, WNT, TGF-b, Hedgehog, and Notch signaling were also enriched.

- Benchmark doses for significantly enriched pathways were lowest at 13 weeks.

- Transcriptional and histological changes at 6 ppm and greater corresponded to dose ranges in which the PK model predicted significant reductions in free GSH and increases in FAcetal.

- Genomic changes at 0.7–2 ppm likely represent changes in extracellular FAcetal and GSH.

- DNA replication stress, enhanced proliferation, squamous metaplasia, and stem cell niche activation appear to be associated with FA carcinogenesis.

- Dose dependencies in MOA, high background FAcetal, and nonlinear FAcetal/GSH tissue kinetics indicate that FA concentrations below 1 or 2 ppm would not increase risk of cancer in the nose or any other tissue or affect FA homeostasis within epithelial cells.

Applicant's summary and conclusion