Dinitrogen tetraoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For a monomer of dinitrogen tetraoxide, nitrogen dioxide, a number of studies reported positive in vitro results in the mutagenicity studies in bacteria and clastogenicity in mammalian cells. Based on this dinitrogen tetraoxide is considered to be genotoxic in vitro.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A monomer of dinitrogen tetraoxide, nitrogen dioxide, was shown to induce both mutagenic and clastogenic effects in several organs following in vivo exposure. Based on this dinitrogen tetraoxide is considered to be genotoxic in vivo.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Mode of Action Analysis / Human Relevance Framework

Dinitrogen tetraoxide exists in equilibrium with its monomer nitrogen dioxide (NO₂), with ca. 25% present in the form of NO₂ at 25 °C. At greater dilution, e.g. with air, the amount of NO₂ is larger. Nitrogen dioxide is an uncharged stable radical and an oxidizing agent. As a free radical, nitrogen dioxide is capable of reacting with biomolecules via different pathways, e.g. abstraction of hydrogen with the formation of nitrous acid and a molecule radical, addition of organic molecules to double bonds, or electron transfer of organic molecules to NO₂ with the formation of a nitrite ion, the biomolecule radical and a proton (MAK, 2005). As a result, NO₂ can produce reactive oxygen species and cause peroxidation of lipid membranes and cell damage. It is most likely that these types of reaction enable its ability to interact with DNA and cause genetic damage in vitro and in vivo. Since this type of reactions is in general non-specific, the observed effects in experimental animals have to be regarded as possibly relevant for humans.

Additional 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 any toxicity studies conducted with either NO₂ or N₂O₄ will share common mechanism. Therefore read-across from NO₂ to N₂O₄ is considered to be justified.

For NO₂ a number of studies reported positive in vitro results in the mutagenicity studies in bacteria and clastogenicity in mammalian cells.Gene mutations were reported in different strains of E. coli and S. typhimurium (Victorin et al., 1998; Kosaka et. al, 1986; Arroyo et al., 1992; MAK (2005) and references quoted therein). Furthermore, increased sister chromatid exchange at a concentration of 5 ppm and chromosomal aberrations at a concentration of 10 ppm in V79 cells after 10 minutes exposure have been reported (Thuda et al, 1981). The induction of DNA strand breaks was also detected in V79 cells after exposure to NO₂concentrations of ≥ 10 ppm for 20 minutes (Görsdorf et al., 1990).

In vivo, no induction of chromosome aberrations was observed in leukocytes and spermatocytes of mice exposed to NO₂ for 6 hours at concentrations of 0.1, 1, 5 and 10 ppm (Gooch et al., 1977). No increase in the number of micronuclei was seen in the bone marrow of mice exposed to NO₂ concentrations of 20 ppm for 23 hours (Victorin et al., 1990). NO₂ also did not induce DNA strand breaks in alveolar macrophages of rats exposed to 1.2 mL/m³ for 3 days (Bermudez et al., 1999).

Dose-dependent increases in mutations and in chromosome aberrations were reported in lung cells from rats exposed to NO₂ for 3 hours at 8, 15, 21 and 28 ppm (15, 29, 40 and 53 mg/m³) (Isomura et al_, 1984). However, this study has several deficiencies, e.g. the survival of the lung cells was very low (10-15%), the conditions of the test were not validated, and the frequency of chromosomal aberrations was determined at a late time point. The animals were not killed until 18 hours after the exposure and the primary lung cells were cultivated for 5 days before they were seeded for the chromosomal aberration test. The preparation and evaluation of the metaphases was carried out three days later. However, this study gives an indication of a possible local genotoxicity of nitrogen dioxide at the port of entry.

Han and co-workers (2013) performed a Comet assay, a micronucleus assay and a DNA-protein complex assay in male rats (6/group) exposed to NO₂ for 6 hours/day for 7 days at analytical concentrations levels of 0, 5, 10 and 20 mg/m³. The testing protocol for the Comet assay had some deviations from the OECD guideline 489. In particular, olive tail moment was used to evaluate DNA damage while % tail DNA is the parameter recommended in the OECD guideline 489. In the Comet assay, single cells suspensions were prepared from brain, lungs, liver, kidneys and spleen following the sacrifice, and 300 cells per group (50/animal, vs. 150/animal recommended by OECD guideline 489) were examined. No information on positive control was provided in the publication. Dose-dependent increases in Olive tail moments (OTM) were observed in cells from all examined organs, except kidneys, where no clear dose-response trend was observed. The observed increases reached statistical significance at all tested dose levels in the lung, liver and kidneys while for brain and spleen cells, statistical significance was reached at two top tested concentrations. In the micronucleus assay, 2000 polychromated erythrocytes from bone marrow were examined from each rat. The frequencies of micronucleated polychromated erythrocytes increased significanty withincreasing concentration of NO₂(1.74, 2.13 and 2.71-fold of control, P < 0.001). The results indicate that NO₂induced the micronuclei formation in a dose dependent manner. Finally, in the DNA-protein complex assay, the DPC coefficients were statistically significantly increased in single cell suspensions from all examined organs (brain, lungs, liver, heart, kidneys, spleen), with statistical significance reached at all dose levels in the liver, at two top dose levels in brain, spleen and heart and at the highest dose level in lungs and kidneys. Based on this results, NO₂ is concluded to induce the formation of DNA-protein complexes under the conditions of the study.

The study of Han et al. (2013) is published in a peer-reviewed journal, sufficient experimental details are provided, and basic scientific principles are met. Because the study shows a clear dose-related increase in the DNA damage in all five tissues analysed, the above mentioned deviations do not affect the scientific acceptability and the study results are considered valid.

Genetic toxicity of the monomer of dinitrogen tetraoxide (N₂O₄), nitrogen dioxide (NO₂), has also been discussed in detail in the reports of MAK (2005) and the Scientific Committee on Occupational Exposure Limits (SCOEL, 2014). The MAK (2005) concluded that in vitro nitrogen dioxide produced clear mutagenic and clastogenic effects, while the study of Isomura et al., 1984, which was considered to be of limited validity, provided some evidence of local genotoxic effects. The SCOEL (2014) evaluation concluded that available studies did not provide evidence of systemic genotoxic effects of nitrogen dioxide, but that further studies may be required to evaluate a possible local genotixicity on epithelia of the airways. It should be noted, however, that neither of these evaluations has taken the study of Han et al. (2013) into consideration.

 

Based on the results of the study of Han and co-workers (2013), the monomer of dinitrogen tetraoxide, nitrogen dioxide, is capable of inducing both mutagenic and clastogenic effectsin vivoin muptiple organs and tissues. As dinitrogen tetraoxide exists in equilibrium with nitrogen dioxide, it is feasible to assume that also dinitrogen tetraoxide is capable of inducing genetic damagein vivo. As dinitrogen tetraoxide is a gas, it is expected to be easily distributed through the body and is therefore capable of reaching gonads. Therefore its ability to cause heritable genotoxic effects cannot be excluded based on the available data.

Justification for classification or non-classification

According to Regulation 1272/2008/EC, the classification of individual substances shall be based on the total weight of evidence available, using expert judgement. The monomer of dinitrogen tetraoxide, nitrogen dioxide, was found to cause both mutagenic and clastogenic effects in vivo in different organs and tissues, including brain, heart, lungs, liver and kidneys. As dinitrogen tetraoxide exists in equilibrium with nitrogen dioxide, it is feasible to assume that also dinitrogen tetraoxide is capable of inducing genetic damage in vivo. As dinitrogen tetraoxide is a gas, it is expected to be easily distributed through the body and is therefore capable of reaching gonads. Thus the substance needs to be considered as having a potential of inducing mutations in germ cells in vivo. Based on this, classification of dinitrogen tetraoxide as Mutagenic Cat. 1B, H340 (May cause genetic defects by inhalation) is proposed in accordance with Regulation 1272/2008/EC.