Dinitrogen tetraoxide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study was performed according to GLP and guideline. This information is read-across from the substance nitrogen dioxide (EC # 233-272-6).

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Eighteen male Wistar rats per test group were whole body exposed to dynamic inhalation atmospheres for 6 hours per working day on consecutive 5 days. The target concentrations were 0.5, 5 and 20 ppm NO2. A concurrent control group was exposed to clean air using the same technical procedures as for test substance exposure.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 27.07.2005 Date of signautre: 21.10.2005

Limit test:

no

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:

- Name of test material (as cited in study report): Nitrogen dioxide
- Physical state: Gas
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: About 7 weeks
- Weight at study initiation: No data
- Fasting period before study: The animals did not have access to water or feed during the exposures
- Housing: During the period when the rats were not exposed they were housed singly in makrolon-wire cages (type MD II, Becker & Co., Castrop-Rauxel, FRG (floor area about 800 cm2)).
- Diet (e.g. ad libitum): Milled mouse/rat laboratory diet "GLP", (Provimi Kliba SA, Kaiseraugst, Basel, Switzerland).
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: 12/13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): A light/dark rhythm of 12 hours was maintained

IN-LIFE DATES: From: To: 18/11/2003 - 05/12/2003

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

The inhalation exposure was carried out considering the following guidelines:

OECD Guidelines for Testing of Chemicals, Section 4: Health Effects, No. 412, "Repeat Dose Inahaltion Toxicity: 28-day or 14-day study"

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration was calculated from the study means of the test pump rates and the supply airflows used during exposure to generate the respective concentrations.

The constancy and the concentrations of the inhalation atmospheres were analysed online by calibrated IR spectrophotometer in all test groups including control.

The analyses were carried out as a separate study at laboratory for inhalation toxicology under the responsibility of Dr. H. Egenolf (principle scientist) of the test facility GKA Analytics, BASF Aktiengesellschaft. The study was carried out in compliance with the principles of GLP.

Duration of treatment / exposure:

6 hours on workdays over a time period of 5 consecutive days.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.88 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
6.4 ppm
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
21.1 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

In Exposure group 1 and Exposure group 2 there were 5 animals per test group.

Control animals:

yes, concurrent vehicle

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

Mortality, clinical observations, bodyweight and determination of pulmonary cell proliferation rate (via Alzet minipums).

Sacrifice and pathology:

Biochemical examinations, lung lavage, cytology, analysis of humoral parameters, necropsy, organ weight, organ sectioning and staining and immunohistology.

Statistics:

Bodyweight: A comparison of each group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.

Lavage parameters except for cell differential analysis (%): Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.

Lipid per-oxidation (MDA-equivalents), 8-HO-Deoxy-guanosine in lung DNA: Pairwise comparison of each dose group with the control group using the WILCOXON-test for the hypothesis of equal means (-sided for; two sided for lipid peroxidation and 8-OH-deoxyguanosine).

Organ weights: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.

Weight parameters, data of S-Phase Responses and Apoptosis: A pairwise comparison of each dose group with the control group was performed using the WILCOXON test (one-sided) for the hypothesis of equal medians.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Exposure to 20 ppm of the test article led to a significant increase of the absolute lung weights.

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

Clinical Observation:
During the whole study period the animals showed no clinical signs and findings different from normal. Concerning the mean body weights there were no statistical significant differences between the exposed animals and the concurrent controls. However, the mean body weight change of the animals exposed to 20 ppm NO2 was slightly lower than the control. Although this difference was not of statistical significance, considering the findings in
the lavage fluid and findings in pathology, this decrease was considered to be of biological relevance, and was probably caused by the irritation in the respiratory tract by the test substance at the high concentration of 20 ppm.

Biochemical examination:
The biochemical parameters malondialdehyde and 8-OH-deoxyguanosine in the lung are indicative for local oxidative stress. There are no biological differences between the test groups and the control in neither of the parameters. However, considering the high standard
deviations, these methods might be not sensitive enough to detect minor changes in in vivo systems.

Clinical Pathology:
In this study broncho-alveolar lavage fluid was analyzed for markers indicative for injury of the bronchi alveolar region, including total protein, activities of lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-glucosaminidase and gamma-glutamyltransferase, as well as differential cell counts.

Increased total protein and elevated activities in gamma-glutamyltransferase and lactate dehydrogenase activities as well as alterations in total cell counts associated with increases in macrophages and polymorphonuclear neutrophils were observed in the broncho-alveolar lavage fluid of the animals exposed to 20 ppm of the test compound. These findings are indicative for a pulmonary irritation and inflammatory potential of the test substance. In the animals exposed to 5 ppm of the test substance only slightly increased gamma-glutamyltransferase activities were noted in the broncho-alveolar fluid. With respect to this finding the no adverse effect level of NO2 was considered to be the low concentration tested (0.5 ppm).

Pathology:
Regarding pathology, inhalation exposure of the test article for 5 days led to significant changes in the lung parenchyma and the trachea. In the high-concentration group of 20 ppm those changes included a significant increase of lung weights, histopathology changes, which were recorded as bronchiolo-alveolar hyperplasia up to severe gradings, mononuclear cell infiltrates, alveolar histiocytosis, and in one case as an alveolar edema. Cell proliferation in the lungs was highly significantly increased with relative increases up to 2328%, when compared with the control (100%). The bronchiolar epithelium (large and medium bronchi and terminal bronchioles) was most severely involved. But also the alveolar epithelium had a slight but significant increase in cell proliferation. The slight significant increase of apoptosis in the large and medium bronchi is regarded to be a reactive response on the clear induction of cell proliferation and so far a secondary (indirect) effect on the exposure with the test article. The tracheal epithelium had a low-grade diffuse hyperplasia as well, which shows that an exposure related toxic impact is also present the upper airways. In the midconcentration group of 5 ppm, the lung weights were in the range of the controls but histopathology still detected a bronchiolo-alveolar hyperplasia in seven of the ten test animals, but less pronounced and to lower gradings. Also the parallel infiltration of mononuclear cells and the extent of alveolar histiocytosis were clearly less pronounced, although still recognizable in some of the exposed animals. In two of the ten test animals, an up to low-grade diffuse hyperplasia of the tracheal epithelium has still been developed as well. Cell proliferation was significantly increased and reached relative values up to 640%,
when compared with the control (100%). The cell proliferation pattern in the lung parenchyma remains comparable with the high concentration group as well, showing the bronchiolar epithelium still as more severely involved, when compared with the alveolar epithelium. In the low-concentration group of 0.5 ppm, the lung weights were in the range of the controls and also histopathology did not exhibit treatment-related effects on the lung parenchyma any longer. The single cases of a low graded alveolar histiocytosis and of few mononuclear cell infiltrates are regarded to be of spontaneous origin, as further histopathology of the lung parenchyma was not present. In addition, semithin and ultrathin sections from the lung of low-concentration group test animals showed no initial, treatment-related alterations after examination by electron microscopy. However, a weak but significant increase of cell proliferation up to a value of 141% was measured in one lung compartment (the medium and large bronchi) of the low concentration group. To clarify, whether the slight increase would have been of more incidental or more treatment related nature, a second set of lung slides were evaluated in the low-concentration group. The evaluation of a second set of slides resulted in slightly lower increased values in the large and medium bronchi of the low concentration group of 0.5 ppm that was not longer of statistical significance. Taking all results of light and electron microscopy and the cell proliferation measurements into consideration, the slight and significant increase of cell proliferation in only one lung compartment (the large and medium bronchi) is regarded to be incidental and still in the normal biological range.

Effect levels

Key result

Dose descriptor:

LOAEC

Effect level:

5 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The exposure of NO2 caused pulmonary irritation in rats at concentrations of 20 and 5 ppm. The animals exposed to a target concentration of 0.5 ppm NO2 were free of findings in clinical pathology and in histopathology. The no effect concentration for NO2 is considered to be approximately 0.5 ppm under the current study condition.