Formaldehyde

Genetic toxicity: in vivo

Administrative data

Endpoint:

genetic toxicity in vivo, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

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

Reference Type:

publication

Title:

Evaluation of inhaled low-dose formaldehyde-induced DNA adducts and DNA–protein cross-links by liquid chromatography–tandem mass spectrometry

Author:

Leng J, Liu CW, Hartwell HJ, Yu R, Lai Y, Bodnar WM, Lu K, Swenberg JA

Year:

2019

Bibliographic source:

Arch Toxicol. 2019 Mar;93(3):763-773

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rats were exposed to 1, 30, and 300 ppb [13CD2]-formaldehyde for 28 days (6 h/day) by nose-only inhalation, followed by measuring DNA mono-adduct (N²-HOMe-dG) and DNA-protein crosslinks (dG-Me-Cys) as formaldehyde specific biomarkers

GLP compliance:

not specified

Type of assay:

other: ultrasensitive nano-liquid chromatography-tandem mass spectrometry

Test material

Test material information

Constituent 1

Reference substance name:

Formaldehyde

EC Number:

200-001-8

EC Name:

Formaldehyde

Cas Number:

50-00-0

Molecular formula:

CH2O

IUPAC Name:

formaldehyde

Details on test material:

- Name of test material (as cited in study report): formaldehyde

Specific details on test material used for the study:

TEST MATERIAL
- Source: Sigma Aldrich, St. Louis, MO, USA
- Purity not specified

Test animals

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Study was conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals
- Age at study initiation: 6 weeks

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

not specified

Details on exposure:

TYPE OF INHALATION EXPOSURE
- Nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- The exposure was done at the Lovelace Respiratory Research Institute (Albuquerque, NM, USA) according to the approved protocols for the use of vertebrate animals in experiments.

TEST ATMOSPHERE
- The concentration of the exposure chamber was monitored by the collection of Waters XpoSure Aldehyde Sampler cartridges every 10 min continuously throughout the exposure

Duration of treatment / exposure:

test group: 28 days, positive control group: 5 days

Frequency of treatment:

6h/day

Post exposure period:

not specified

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 other: ppb

Doses / concentrations 2

Dose / conc.:

30 other: ppb

Doses / concentrations 3

Dose / conc.:

300 other: ppb

No. of animals per sex per dose:

not specified

Control animals:

yes

Positive control(s):

Rats that served as the positive control samples were exposed to 10 ppm [13CD2]-FA for 5 days (6 h/day). The formation of exogenous formaldehyde-induced DNA adducts in these 10 ppm exposed samples have been well validated in a previous study (Lu et al. 2010)

Examinations

Tissues and cell types examined:

nasal mucosa, bone marrow, PBMC, trachea, liver, hippocampus, olfactory bulb, cerebellum, lung

Details of tissue and slide preparation:

Animals were euthanized with a pentobarbital-based euthanasia solution to induce surgical level anesthesia and pneumothorax was introduced. Tissue samples were harvested, wrapped in aluminum foil, and immediately snap frozen in liquid nitrogen. They were stored at -80 °C until DNA isolation, reduction, digestion, HPLC purification, and adducts quantitation by nano-LC–MS–MS. These standard procedures have been extensively used in previous studies (Lu et al. 2010, 2011, 2012; Yu et al. 2015; Lai et al. 2016; Liu et al. 2018).

Evaluation criteria:

not applicable

Statistics:

Analysis of variance (ANOVA) was used to compare the means of formaldehyde-induced DNA adducts and DPCS across the control and exposure groups.

Results and discussion

Test results

Sex:

male

Genotoxicity:

not determined

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Exposure caused no adverse clinical observations or gross pathology.
After exposure to 10 ppm for 6/day over 5 days the endogenous and exogenous N²-
HOMe-dG were both detected and quantified with the amounts of 3.41 and 2.70 per 107 dG, respectively. Likewise, the endogenous and exogenous dG-Me-Cys were detected and quantified with the amounts of 3.67 and 2.47 per 108 dG, respectively. These results have demonstrated that the methods are capable to detect both endogenous and exogenous formaldehyde-induced DNA adducts or DPCs. These methods are highly sensitive, accurate and precise as shown
by our extensive validation.
N²-HOMe-dG: After exposure to 1, 30 and 300 ppb for 6h/day over 28 days endogenous formaldehyde DNA adducts were all detected, but no exogenous formaldehyde DNA adducts could be found in any groups. Likewise, endogenous adducts were detectable in all other tissues distant to nasal epithelium, with the levels ranging from 2.35 to 5.25 adducts per 107 dG, depending on the specific tissue types. No exogenous formaldehyde-induced N2-HOMe-dG was detected in any tissues analyzed.
dG-Me-Cys: After exposure to 1, 30 and 300 ppb for 6h/day over 28 days no exogenous formaldehyde DPC was found in nasal epithelium of rats exposed to formaldehyde, while endogenous formaldehyde adduct was detected in all samples. Besides, endogenous formaldehyde induced dG-Me-Cys was also detected in all tissues distant to nasal epithelium, with 1.52 to 8.03 adducts per 108 dG across different tissues. No exogenous formaldehyde-induced dG-Me-Cys was detected in any analyzed tissues.
Since no N²-HOMe-dG and dG-Me-Cys caused by exogenous FA was detected in any tissues, even at the highest exposure level of 300 ppb in this study, it indicates that the levels of DNA adducts were below the limit of detection. Typically, DNA amount from each rat nasal epithelium is small (10–20 μg), which could limit our capability of detecting these extremely low abundant DNA adduct following a low-dose exposure. To address this, we pooled five nasal samples exposed to 300 ppb FA and still no exogenous N²-HOMe-dG was found further illustrating that the levels of exogenous N²-HOMe-dG were below the limit of detection when exposed to low dose of FA at 300 ppb or lower.
No statistical significant difference was found for either mono-adduct or DPCs in any tissues, indicating that exogenous FA did not alter the endogenous adducts levels at 300 ppb or lower. The concentrations of dG-Me-Cys in the liver were relatively higher than the other tissues examined.

Applicant's summary and conclusion

Conclusions:

Endogenous adducts are present in all tissues analyzed, but exogenous adducts were not detectable in any tissue samples, including the most susceptible nasal
epithelium. Moreover, formaldehyde exposure at 1, 30 and 300 ppb did not alter the levels of endogenous formaldehyde induced DNA adducts or DNA-protein crosslinks.

Executive summary:

In a reliable study inhaled low-dose formaldehyde-induced DNA adducts
and DNA–protein cross-links were evaluated by liquid chromatography–tandem mass spectrometry. Rats were exposed to 1, 30, and 300 ppb [13CD2]-formaldehyde for 28 days (6 h/day) by nose-only inhalation, followed by measuring DNA mono-adduct (N²-HOMe-dG) and DNA–protein crosslinks (dG-Me-Cys) as formaldehyde specific biomarkers. Both exogenous and endogenous DNA mono-adducts and dG-Me-Cys were examined with ultrasensitive nano-liquid chromatography–tandem mass spectrometry. The data clearly show that endogenous adducts are present in all tissues analyzed, but exogenous adducts were not detectable in any tissue samples, including the most susceptible nasal
epithelium. Moreover, formaldehyde exposure at 1, 30 and 300 ppb did not alter the levels of endogenous formaldehyde-induced DNA adducts or DNA-protein crosslinks. The novel findings from this study provide new data for risk assessment of exposure to low doses of formaldehyde.