Dinitrogen tetraoxide

Endpoint summary

Description of key information

Based on read-across from a monomer of dinitrogen tetraoxide, NO2, the substance causes adverse effects in lungs after respiratory exposure. In the available 90 -day repeated dose toxicity study performed according to modern standards, no adverse systemic or local effects were observed at the highest tested dose of 2.5 ppm. SCOEL (2014) concluded that the level of 0.5 ppm (0.955 mg/m3) as 8 -h TWA is considered sufficient to protect against adverse respiratory effects by repeated exposure. As dinitrogen tetraoxide represents a dimer of nitrogen dioxide, one mole of dinitrogen tetraoxide will generate two moles of nitrogen dioxide. Therefore based on molar ratio for dinitrogen tetraoxide the 8-h TWA IOEL was recalculated to 0.25 ppm by the registrant, which is equal to 0.955 mg/m3. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

4.36 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study has a Klimisch score 2 because it is a read-across study.

System:

other: no systemic effects observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

4.36 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study has a Klimisch score 2 because it is a read-across study.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Dinitrogen tetraoxide exists in equilibrium with nitrogen dioxide, with ca. 25% present in the form of NO₂at 25 °C. At greater dilution, e.g. with air, the amount of NO₂is 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 NO₂to its dimer N₂O₄is considered to be justified. 

NO₂ is a moderate oxidation agent and an uncharged, stable free radical capable of initiating radical reactions and producing reactive oxygen species.NO₂ reacts slowly in water to form nitric acid and nitrous acid, both of which have irritative to corrosive effects.In addition there may be damage to the tissue in the terminal airways caused by radical reactions of NO₂ with components of the alveolar fluid and the epithelial cells. Also inflammatory symptoms are observed. After long-term exposure emphysema-like changes develop, with changed lung function parameters, in particular an increased residual lung volume and increased expiratory resistance.

As these types of reactions are generally non-specific, the observed effects in experimental animals need to be considered as relevant for humans.

Additional information

Repeated dose toxicity of the monomer of dinitrogen tetraoxide (N₂O₄), nitrogen dioxide (NO₂), has been discussed in detail in the reports of MAK (2005) and Scientific Committee on Occupational Exposure Limits (SCOEL, 2014). 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. 

 

As NO₂ is a gas, the main route of exposure is via inhalation. The target organs of NO₂ are terminal airways. NO₂ is a moderate oxidation agent and an uncharged, stable free radical capable of initiating radical reactions and producing reactive oxygen species. NO₂ reacts slowly in water to form nitric acid and nitrous acid, both of which have irritative to corrosive effects. In addition there may be damage to the tissue in the terminal airways caused by radical reactions of NO₂ with components of the alveolar fluid and the epithelial cells. Also inflammatory symptoms are observed. After long-term exposure emphysema-like changes develop, with changed lung function parameters, in particular an increased residual lung volume and increased expiratory resistance.

 

The number of repeated dose toxicity studies with either N₂O₄ or NO₂ compliant with modern guidelines is very limited. One reliable 90-day toxicity study with rats was conducted in 2006 by BASF. Groups of 15 male and 10 female Wistar rats were exposed to NO₂in whole-body exposure chambers at analytical concentrations of 0.008, 0.25, 0.82 and 2.15 ppm during 5 days/week for 6 hours/day for 13 weeks. No substance-induced effects, either local or systemic, were observed up to the highest concentration level of 2.15 ppm. Therefore, the authors specified a NOAEC of 2.15 ppm for this study. However, in the dose-range finding study, in which18 male Wistar rats per group were exposed to NO₂in whole-body exposure chambers during 5 days for 6 hours/day at analytical concentrations of 0, 0.1, 4.9 and 19.2 ppm,histopathological alterations in the lungs (bronchoalveolar hyperplasia, mononuclear cell infiltration and alveolar histiocitosis) and in the trachea (diffuse hyperplasia of the tracheal epithelium) and increased cell proliferation (up to 640% of the control) in the large and medium bronchi, terminal bronchioles and in the alveoli occurred at 5 ppm and above. Significantly increased lung weights, alveolar oedema, increased apoptosis rates in the medium and large bronchi as well as increased cell numbers, an increased number of macrophages and polymorphonuclear neutrophils, increased total protein levels and an increased activity ofγ-glutamyltransferase and lactate dehydrogenase in the BALF were additionally found at 20 ppm. The value of 5 ppm was considered to be a LOAEC.

 

There are reports that continuous long-term exposure (23 to 24 hours a day) of experimental animals to NO₂ concentrations of 5 ppm and above caused emphysema, as is also observed in the human lung. Sagai et al. (1984) and Kubota et al. (1987) (studies quoted in MAK (2005) and SCOEL (2014) evaluations) reported the exposure of male rats continuously (24 hours/day) to 0, 0.04, 0.4 and 4 ppm NO₂ for 9, 18 or 27 months in whole-body exposure chambers. In rats exposed to NO₂concentrations of 0.04 ppm continuously for 27 months, evidence of lipid peroxidation was observed, as demonstrated by increased ethane exhalation, and after exposure to 0.4 ppm and above, histopathological effects on the lungs were observed. From 0.4 ppm, the mean thickness of the air-blood barrier was increased slightly after 18 months and significantly after 27 months. Moreover, there was some interstitial edema and slight bronchiolar and alveolar epithelium changes. Hypertrophy and hyperplasia of the bronchiolar epithelium, Clara cell hyperplasia, interstitial fibrosis and type I and type II cell hypertrophy were observed at 4 ppm after 27 months exposure. The SCOEL (2014) evaluation estimated that if the exposure times were converted from 24 hours to 8 hours exposure duration, it would theoretically correspond to the LOAEC of 1.2 ppm, with severe effects (fibrosis) observed at ca. 12 ppm.

 

Based on the available human exposure data obtained in working populations, the SCOEL (2014) recommended the value of 0.5 ppm (0.955 mg/m³) as the 8-h TWA Occupational Exposure Limit for worker exposure (IOEL). The evaluation of the SCOEL (2014) concluded that this level is considered to be safe with respect to adverse effects on the lung function under conditions of chronic occupational exposure. Furthermore, it is concluded in the SCOEL report that available data from experimental animal studies are in full support of the IOEL of 0.5 ppm. As dinitrogen tetraoxide represents a dimer of nitrogen dioxide, one mole of dinitrogen tetraoxide will generate two moles of nitrogen dioxide. Therefore based on molar ratio for dinitrogen tetraoxide this value was recalculated to 0.25 ppm by the registrant, which is equal to 0.955 mg/m³.

 

As NO₂ is capable of reacting with water forming nitric and nitrous acids, which both have corrosive properties, for classification and labelling purposes it needs to be evaluated whether the adverse effect is a reflection of true repeated exposure toxicity or whether it is in fact a local (corrosive) effect. According to the ECHA Guidance on the application of CLP criteria (2015), one way to distinguish between these possibilities is to consider the dose level which causes the toxicity. If the dose is more than half an order of magnitude lower than that mediating the evident acute toxicity (corrosivity) then it could be considered to be a repeated-dose effect distinct from the acute toxicity. In this case, classification as specific target organ toxicant (repeated exposure) would be warranted even if the substance (or mixture) is also classified as acutely toxic and/or corrosive. In case of NO₂, occurrence of lung emphysema, hypertrophy and hyperplasia of bronchial epithelium and interstitial fibrosis were reported to occur at concentration levels ≥ 4 ppm; however, as these studies involved continuous (23-24 hours) exposure, they are considered not suitable for classification and labelling purposes. In a 5-day dose range finding study following 6-hour/day, 5-day/week exposure, adverse effects on the lung (alveolar hystiocytosis, bronchoalveolar hyperplasia, mononuclear cell infiltration and increased cell proliferation) occurred at 5 ppm. In the available acute inhalation toxicity study with rats (Carson, 1962) 1-hour LC50 was 115 ppm. The extrapolation to 4-hour exposure duration using Haber’s law, as recommended by ECHA Guidance on data requirements and chemical safety assessment, would result in 4-hour LC50 of ca. 28 ppm. One hour exposure to 28 ppm in the same study resulted in only mild signs of nasal irritation in rats, with no changes in the lung to body weight ratio. No gross pathological lesions were noted. One hour exposure to 72 ppm caused signs of severe respiratory distress and eye irritation in rats lasting about 2 days but no deaths. The lung to body weight ratios significantly increased during the first 48 h after exposure but in most cases returned to normal by the seventh day. Thus evident acute toxicity effects occur after one hour exposure to 72 ppm, which corresponds to 18 ppm for 4-hour exposure duration using Haber’s law. The effects in the repeated dose toxicity dose-range finding study were observed at the exposure concentration of 5 ppm, which is less than half an order of magnitude lower than the concentration at which clear signs of acute toxicity were observed in the acute inhalation toxicity study. It should also be noted that in the 90-day repeated dose toxicity study no adverse effects were observed at the highest tested concentration of 2.5 ppm. Therefore the adverse effects observed in the dose-range finding study are considered to be a result of a corrosive nature of the substance and are therefore covered by the classification of the substance as Acute Tox. 1 and Skin Corr. 1B. Therefore no classification of dinitrogen tetraoxide for repeated dose toxicity is considered to be warranted in accordance with Regulation 1272/2008/EC.    

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The substance is a gas and therefore the inhalation route is considered to be the most appropriate route of exposure. A 28 or 90 day repeat dose oral toxicity study therefore does not need to be conducted.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
This study was performed according to GLP and an appropriate guideline.

This information is read-across from the substance nitrogen dioxide (EC # 233-272-6).

Justification:
It is understood that there is an equilibrium existing between NO2 and N2O4.Hence, it would seem inevitable that any toxicity studies conducted with these oxides will share common toxicities. It also seems plausible that the prevalent oxide of nitrogen would be Nitrogen Dioxide.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
This study was performed according to GLP and an appropriate guideline.

This information is read-across from the substance nitrogen dioxide (EC # 233-272-6).

Justification:
It is understood that there is an equilibrium existing between NO2 and N2O4. Hence, it would seem inevitable that any toxicity studies conducted with these oxides will share common toxicities. It also seems plausible that the prevalent oxide of nitrogen would be Nitrogen Dioxide.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The substance is a gas and therefore the inhalation route is considered to be the most appropriate route of exposure. A 28 or 90 day repeat dose dermal toxicity study therefore does not need to be conducted.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
The substance is a gas and therefore the inhalation route is considered to be the most appropriate route of exposure. A 28 or 90 day repeat dose dermal toxicity study therefore does not need to be conducted.

Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: lung

Justification for classification or non-classification

Based on the evaluation of the available data, it is concluded that effects seen in repeated dose toxicity studies with the monomer of dinitrogen tetraoxide, nitrogen dioxide, are the reflection of the corrosive nature of the substance. These effects are therefore covered by the classification of the substance as Acute Tox. 1 and Skin Corr. 1B. Therefore classification of dinitrogen tetraoxide for repeated dose toxicity is considered to be not warranted in accordance with Regulation 1272/2008/EC.