Nitrogen monoxide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The report’s conclusion is confirmed: no chromosome dammage or bone marrow cell toxicity in vivo on male rats.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Nitrogen monoxide

EC Number:

233-271-0

EC Name:

Nitrogen monoxide

Cas Number:

10102-43-9

Molecular formula:

NO

IUPAC Name:

oxidoamine

Specific details on test material used for the study:

The tested batch contained 900 ppm NO in nitrogen; tested concentrations are expressed as pure NO

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

The preliminary study with males and females evidenced an absence of difference in sensitivity between sexes
Animals were weighing between 140 and 150 grams.
Animals were acclimatised for a minimum of 5 days, examined daily and weighed prior to dosing.

ENVIRONMENTAL CONDITIONS :
Temperature : 21 +/- 2 °C
Relative humidity : 55 +/- 15%
Light : artificial light for 12 hours per day
Food and water ad libitum

Administration / exposure

Route of administration:

inhalation: aerosol

Vehicle:

Air

Details on exposure:

Snout only inhalation exposure

Duration of treatment / exposure:

6 hours of exposure

Frequency of treatment:

1

Post exposure period:

24 or 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 ppm

Doses / concentrations 2

Dose / conc.:

75 ppm

Doses / concentrations 3

Dose / conc.:

150 ppm

Remarks:

Corresponds approximately to the MTD

No. of animals per sex per dose:

30 ppm : 7 rats
75 ppm : 7 rats
150 ppm : 14 rats

Negative control : 14 rats
Positive control : 5 rats

Control animals:

yes

yes, concurrent no treatment

Positive control(s):

Cyclophosphamide (batch number : 108H0568). It was prepared as a solution in purified water at a concentration of 2 mg/ml just prior to administration. It was administrated orally by gastric intubation at a volume dosage of 10ml/kg bw.

Examinations

Tissues and cell types examined:

Femurs are dissected out from each animals. The femurs were cleared of tissue and the proximal apiphys removed from each bone.

Details of tissue and slide preparation:

Due to the presence of mast cell granules in rat bone smears, which appear identical to micronuclei when stained using the Romanowsky methods, a modified Feulgen staining method is employed for the rat micronucleus test in this laboratory. This method specifically stains DNA-containing bodies deep purple while leaving mast cell granules unstained. The method also allows reasonable differentiotion of mature and immature erythrocytes and produces permanent preparations.
The slides were fixed and stained as described in the following schedule :
1/ Fixed for 10 minutes in methanol
2/ Hydrolysed in Bouin's fluid at room temperatur for 30 hours
3/ Washed three times in purified water (5 minutes per wash)
4/ Stained in Schiff's reagent for one hour at room temperature
5/ Washed three times in purified water
6/ Counter-stained for ten minutes in very dilute aqueous Eosin yellowish
7/ Washed for five minutes in purified water
8/ Stained for 30 minutes in Mayer's Haemalum diluted 9 volumes : 1 volume with aqueous acridine orange solution in purified water (1 mg/ml)
9/ Rinsed in purified water
10/ Rinsed in running tap water
11/ Washed for 5 minutes in purified water
12/ Air dried
13/ Slides were mounted with coverslips using DPX mountant
14/ The mountant was allowed to harden

The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. Usually only one smear per animals was examined. The remaining smears were held temporarily in reserve in case of technical problems with the first smeat.

Evaluation criteria:

Micronuclei are identified by the following criteria:
- Large enough to discern morphological characteristics
- Should possess a generally rounded shape with a clearly defined outline
- Should be deeply stained and similar in colour to the nuclei of other cells - not black
- Should lie in the same focal plane as the cell
- Lack internal structure, ie they are pyknotic
- There should be no micronucleus-like debris in the area surrounding the cell

The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during assessment of this proportion was also kept.

Results for each treatment group were compared with the results for the concurrent control group using non-parametrics statistics.

A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (P <0.01); individual and/ or group mean values should exceed the laboratory historical control range (Morrison and Ashby 1995). A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (P >0.01) and where these values fall within the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.
Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant dose-related decrease in the proportion of immature erythrocytes (P<0.01).

Statistics:

Non-parametric statistical methods were chosen for analysis of results because:
• They are suited to analysis of data consisting of discrete/ integer values with ties such as the incidence of micronucleated immature erythrocytes
• The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of non-parametric analysis and classical analysis of variance are very similar)
• “Outliers” are frequently found in the proportion of immature erythrocytes for both control and treated animals; non-parametric analysis based on rank does not give these values an undue weighting.

For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts). Comparison of several dose levels is made with the concurrent control using the Linear by Linear Association test for trend, in a step-down fashion if significance is detected (Agrestu et al. 1990); for individual intergroup comparisons (ie the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at 150 ppm 2 males were found dead after 6 hours of exposure. At post mortem examination, one animal showed no abnormalities and the other moderate congestion in all lungs lobes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

For more details, please refer to the illustration summing results below

Applicant's summary and conclusion

Conclusions:

In a OECD 474 study, NO did not induce micronuclei in erythrocytes, and was therefore non genotoxic, in rats exposed for 48h by inhalation up to 150 ppm (exceeding the maximum tolerated dose).

Executive summary:

No statistically significant increase in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in rats treated with nitric oxide and killed 24 or 48 hours later, compared to vehicle control values (P>0.01 in each case).

It is concluded that nitric oxide did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administred by snout only inhalation exposure in this in vivo test procedure.