Dinitrogen tetraoxide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Remarks:

data published

Adequacy of study:

key study

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Data published, no original study report available

Justification for type of information:

Dinitrogen tetraoxide exists in equilibrium with nitrogen dioxide, with ca. 25% present in the form of NO2 at 25 °C. At greater dilution, e. g. with air, the amount of NO2 is greater. Therefore it would seem inevitable that the toxicity studies conducted with either of these gases would share a common mechanism. Therefore read-across from NO2 to its dimer N2O4 is considered to be justified.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: groups of 6 male rats were exposed to the test substance at concentrations 5, 10 and 20 mg/m3 for 6 h/d for 7 days. 18 h after the last exposure rats were sacrificed and brain, lung, liver, spleen, heart and kidney collected from the individual rat to prepare single cell suspensions. The protein-DNA complexes were precipitated with sodium dodecyl suphate/KCl, resuspended with proteinase K and centrifuged to collect supernatant (protein-bound DNA). Fluorescence was measured at an excitation wave length of 350 nm and an emission wave length of 450 nm. The result was expressed as the percentage of protein-bound DNA to total DNA (free DNA plus protein-bound DNA).
- Short description of test conditions: rats were exposed whole-body in inhalation chambers at concentrations 5, 10 and 20 mg/m3
- Parameters analysed / observed: fluorescence

GLP compliance:

not specified

Type of assay:

other: DNA-protein crosslinks assay

Test material

Test material information

Constituent 1

Reference substance name:

Nitrogen dioxide

EC Number:

233-272-6

EC Name:

Nitrogen dioxide

Cas Number:

10102-44-0

IUPAC Name:

nitrous acid

Test material form:

gas

Details on test material:

Supplier: Beijing Beifen-Ruili Analytical Instrument (Group) Co., Ltd. (Beijing, China);

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Center of Experimental Animal of Hebei Province, China
- Age at study initiation:ca. 3 months old
- Weight at study initiation: 280-300 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: stainless steel cages (50 x 40 x 25 cm); number of animals per cage not specified
- Diet: ad libitum, except during the treatment period
- Water: ad libitum, except during the treatment period
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 24 ± 2
- Humidity (%): 50 ± 5%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: not specified

Administration / exposure

Route of administration:

inhalation: gas

Vehicle:

None

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 m3 exposure chambers containing continuous concentrations of 5, 10 and 20 mg/m^3 (2.66, 5.32 and 10.64 ppm) NO2
- Method of holding animals in test chamber: not restrained
- Source and rate of air: fresh air, not specified further. The NO2 gas was diluted with fresh air at the intake port of the chamber to yield desired concentrations; then, it was delivered to the animals through a tube positioned in the upper part of each chamber and distributed homogeneously via a fan.
- Method of conditioning air: not specified
- Temperature, humidity, pressure in air chamber: not specified
- Air flow rate: not specified
- Air change rate: not specified
- Method of particle size determination: not applicable, the substance is a gas
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: The NO2 concentration within the chambers was measured every 30 min by a Saltzman colorimetric method using a spectrometer calibrated at 540 nm
- Samples taken from breathing zone: no

Duration of treatment / exposure:

6 hours/day, 7 days

Frequency of treatment:

Daily

Post exposure period:

18 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air (analytical)

Remarks:

Concurrent air controls

Doses / concentrations 2

Dose / conc.:

5 mg/m³ air (analytical)

Remarks:

Equal to 2.66 ppm

Doses / concentrations 3

Dose / conc.:

10 mg/m³ air (analytical)

Remarks:

Equal to 5.32 ppm

Doses / concentrations 4

Dose / conc.:

20 mg/m³ air (analytical)

Remarks:

Equal to 10.64 ppm

No. of animals per sex per dose:

6 males/dose

Control animals:

yes, sham-exposed

Positive control(s):

Not used.

Examinations

Tissues and cell types examined:

Brain, lung, liver, spleen, heart and kidney

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: not specified

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): sacrifice 18 hours post last exposure

DETAILS OF SLIDE PREPARATION: Isolated organs were washed with ice-cold phosphate-buffered saline (PBS), and were forced through a wire-mesh screen to obtain 1.5 x 10^6 mL single-cell suspension. The suspension was treated with sodium dodecyl sulfate (SDS, 2%) at 65 °C for 10 min, then KCl (1 M in 10 mM Tris–HCl, pH 7.4) was added into the system by carefully mixing, followed by passing the mixture
through a 1 mL polypropylene pipette tip for six times to shear DNA to a uniform length. The sample was cooled on ice for 5 min and centrifuged at 10,000 rpm for 5 min at 4 °C. The supernatant containing the unbound fraction of DNA (free DNA) was collected in the other tube. The SDS-K+ precipitate containing the protein and DPC complexes was re-suspended with wash buffer (0.1 M KCl, 0.1 mM EDTA, 20 mM Tris–
HCl, pH 7.4) at 65 °C for 10 min, cooled on ice for 5 min, and centrifuged as mentioned above. The precipitate was washed for three times, and supernatant from each wash step was pooled with previous unbound DNA fraction. The final pellet was re-suspended in wash buffer with proteinase K at 50 °C for 3 h, then the digest was placed on ice for 5 min and centrifuged at 12,000 rpm (4 °C) for 10 min to collect supernatant protein-bound DNA). Hoechst 33,258 was added to each supernatant (containing free DNA and protein-bound DNA, respectively) obtained from previous steps in the dark for 30 min.

METHOD OF ANALYSIS: Fluorescence was measured at an excitation wave length of 350 nm and an emission wave length of 450 nm. The result was expressed as the percentage of protein-bound DNA to total DNA (free DNA plus protein-bound DNA).

Evaluation criteria:

Statistically significant increase in percentage of protein-bound DNA to total DNA (free DNA plus protein-bound DNA) was considered to be a positive result.

Statistics:

ANOVA was applied for between-group statistical comparison using Origin 7.0 software. Differences were considered significant when P < 0.05, P < 0.01, P < 0.001.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Remarks:

no mortality/morbidity, no efects on the mean body weight gain, no visual changes at necropsy

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

RESULTS OF DEFINITIVE STUDY

NO2 inhalation caused a statistically significant increase of DPC formation in a dose-dependent manner in all organic cells. For brain, spleen and heart cells, DPC coefficients tended to increase at 5 mg/m3 NO2, but no statistically significant differences were observed (P > 0.05), whereas statistically significant increase was observed after 10 and 20 mg/m3 NO2 exposure (mean values 1.17-, 1.32-fold of control for brain, 1.61-, 1.68- fold of control for spleen and 1.42-, 1.69-fold of control for heart, respectively, P < 0.05). For lung and kidney cells, lower concentrations (5 and 10 mg/m3) of NO2 tended to increase DPC coefficient, but no statistically significant differences were observed (P > 0.05); whereas, 20 mg/m3 NO2 exposure statistically significantly enhanced the level of DPC (1.62- and 1.71-fold of control, respectively, P < 0.05). For liver cells, DPC coefficients were statistically elevated after NO2 treatment at all concentrations tested, and the difference amplified with the increase of inhaled concentration (1.27-fold of control for 5 mg/m3, P < 0.05; 1.73-fold of control for 10 mg/m3, P < 0.01; 1.99-fold of control for 20 mg/m3, P < 0.001).

Applicant's summary and conclusion

Conclusions:

In the DNA-protein complex (DPC) assay, conducted in combination with the Comet and micronucleus assays, nitrogen dioxide caused a statistically significant increase of DPC coefficients in a concentration-dependent manner in all investigated cells (brain, lung, liver, kidney, spleen and heart).

Executive summary:

In the DNA-protein complex (DPC) assay, conducted in combination with a Comet and micronucleus assays, groups of 6 male Wistar rats were exposed whole body to 0 (concurrent air controls), 5, 10 and 20 mg NO₂/m³ for 6 hours/day for 7 days. Positive controls were not included. There was no toxicity. Rats were sacrificed 18 hours after the last exposure, and brain, lung, liver, spleen, heart and kidney were collected from the individual rat to prepare single-cell suspensions. The DPC coefficients (percentage of protein-bound DNA to total DNA) were determined by spectrophotofluorimetry at an excitation wave length of 350 nm and an emission wave length of 450 nm. NO₂ inhalation caused a statistically significant increase of DPC formation in a dose-response manner in all organic cells. For brain, spleen and heart cells, statistically significant increases were observed at 10 and 20 mg/m³ NO₂ exposure in a dose-dependent manner, while for lung and kidney cells, statistically significant increases were noted at the highest concentration of 20 mg/m³ NO₂. For liver cells, DPC coefficient was statistically elevated after NO₂ treatment at all concentrations tested in a dose-dependent manner. Based on this results, NO₂ is concluded to induce the formation of DNA-protein complexes under the conditions of the study.