Dinitrogen tetraoxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Chromosomal aberrations and sister-chromatid exchanges induced by gaseous nitrogen dioxide in cultured Chinese hamster cells

Author:

Tsuda H, Kushi A, Yoshida D & Goto F

Year:

1981

Bibliographic source:

Mutation Research; 89, 303-309

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Effects of gaseous nitrogen dioxide (NO2) on chromosomal morphology of cultured Chinese hamster Y79-H3 cells were investigated. Chinese hamster cells were exposed to NO2 gas in N2 gas at NO2 concentrations of 0, 5, 10, 20, 50 and 100 ppm (v/v) for 10 min at a gas flow rate of 1000 ml/min. Both chromosomal aberrations and sister-chromatid exchanges were increased depending on the NO2 concentration.

GLP compliance:

no

Remarks:

Study would have predated GLP

Type of assay:

in vitro mammalian cell micronucleus test

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 (NO2)
- Physical appearance: gas
- Purity: not indicated
- Batch No.: not indicated
- Supplier: Takachicho Co., Japan

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Test concentrations with justification for top dose:

0, 5, 10, 20, 50 and 100 ppm (v/v)

Details on test system and experimental conditions:

NO2 gas treatment:
V79-H3 cells were seeded into a 60 mm dish at a density of 2.5 or 5 x 10^5 cells. 24 h after seeding, the cells were washed 3 times with Hanks balanced salt solution. After the washing the dishes were put, updside down into the gas-exposure apparatus.
The NO2-N mixture was then allowed to flow into the apparatus continuously for 10 min at a flow rate of 1000 ml/min at room temperature (25°C). A longer exposure time resulted in serious drying og the cells under photo-microscopial observation. Each NO2 concentration was obtained by dilution of the original NO2 gas (100 ppm in N2) with N2 gas. The NO2 concentrations were monitored chemiluminescently by NOx analyzer. The inner volume of the gas exposure apparatus was 300 ml. Within 1 min, the NO2 concentration reached a desirable concentration.

Chromosmal aberration test:
Immediately after the treatment with NO2 gas the cells were washed twice with Hanks salt solution and maintained in normal culture medium for 20 h. For the last 3 h colcemid was added at 0.1µg/ml. To establish each point 100 metaphase cells were scored.

Sister chromatid exchange:
Immediately after the treatment with NO2 gas the cells were washed twice with Hanks salt solution and maintained in culture medium contatining 10µM of bromodeoxyuridine for 44 h. For the last 3 h colcemid was added at 0.1µg/ml. To establish each point 30 metaphase cells were scored.

Measurement of NO2 and water adhering to the dish surfaces
Immediately after exposure to the NO2 gas, NO2 adhering to the dish surface was collected by washing 3 times with Griess-Salzman reagent and its content was measured colorimetrically. Water adhering to the dish surface was measured by weight. The NO2 concentration on the dish surface was calculated from NO2 and water contents

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

all strains/cell types tested

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

increased frequency of chromosomal aberrations and chromatid exchanges

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Chromosomal aberrations by NO2 gas:
The percentages of metaphase cells with one or more chromosomal aberrations were increased with increasing concentrations of NO2 (Table 1). At 100 ppm, NO2 induced very significant aberrations (35%) including typical chromatid breaks and chromatid exchanges. Even at lower concentration (10 ¿ 50 ppm) chromosal aberrations were induced significantly, the main aberrations being chromatid gaps and breaks.

Sister chromatid exchanges by NO2 gas
As shown in table 2, NO2 gas increased sister chromatid exchanges per metaphase significantly at NO2 concentrations of 5, 10, 20, 50 and 100 ppm with enhancing factors of 1.3, 1.7, 1.6, 2.2 and 2.5 respectively.

NO2 adhering to the dish surfaces
Actual amounts were 4.7µg/dish at 100 ppm NO2, 1.8µg/dish at 50 ppm NO2and 0.6µg/dish at 10 ppm NO2. The amount of Hanks salt solution adhering to the dish surface was 0.15 ml/dish after exposure to any concentration of NO2gas for 10 min. Therefore the concentrations of NO2on the dish surfaces were 0.66mM at 100 ppm NO2, 0.26 mM at 50 ppm NO2 and 0.08mM at 10 ppm NO2, respectively. The pH of the Hanks salt solution on the dish surface remained almost unchanged (pH 7.2-7.6) even after exposure to NO2 at 100 ppm.

Any other information on results incl. tables

Table 1: Chromosomal aberrations induced by gaseous NO₂ in cultured Chinese hamster V79 -H3 cells

NO2 (ppm)	Abnormal^ametaphases (%)	Aberrations per 100 metaphases
		Chromatid gaps	Chromosome gaps	Chromatid breaks	Chromosome breaks	Chromatid exchanges	Dicentric chromosomes	Ring chromosomes	Minutes
No treatment	2	2	0	0	0	0	0	0	0
0	6	4	0	2	0	0	0	1	0
5	9	7	0	2	0	1	0	0	0
10	10	8	1	1	0	0	0	0	0
20	15	7	1	6	0	0	1	0	1
50	24	10	3	9	0	0	2	0	5
100	35	23	3	41	1	17	1	1	21
100^b	6	3	1	2	0	0	0	0	0

^aTo establish each point, 100 metaphases cells were exposed

^bCells were not washed before exposure to NO₂gas

Table 2: Sister chromatid exchanges induced by gaseous NO₂ in cultured Chinese hamster V79 -H3 cells

NO2(ppm)	SCE per^ametaphase	SE^b	Range	Enhancing factor
No treatment	5.6	0.32	3-9	1
0	5.8	0.42	2-11	1.04
5	7.0	0.51	3-13	1.25
10	9.3	0.47	3-15	1.66
20	9.0	0.45	5-17	1.61
50	12.4	0.63	7-22	2.21
100	13.7	0.71	9-25	2.45

^aSister chromatid exchanges per metaphase cell. To establish each point, 30 metaphase cells were scored

^bStandard error

Applicant's summary and conclusion

Conclusions:

The results showed that gaseous NO2 increased the incidence of both chromosomal aberrations and sister chromatid exchanges in cultured Chinesehamster V79-H3 cells at NO2 concentrations of 10, 20, 50 and 100 ppm for 10 min.