Nitrogen monoxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

No noteworthy deviation (test had to be adapted to a gas). The report’s conclusion is confirmed: mutagenic at non-cytotoxic

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

1/ plates from the first range-finder experiment were incubated for a period of 4 days post treatment (rather than 2-3 days); 2/ Due to delays in test article supply and method development, the schedules details in the protocol were altered.

Principles of method if other than guideline:

Nitric oxide was tested in 4 strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and 2 strains of Escerichia coli (WP2 pKM101 and WP2 uvrA pKM101) in triplicate, with and without S9 mix, at concentrations set after a range finding assay. Negative (solvent) were included in both assays, in quintuplicate without and with S9. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S9. The activity of S9 mix used in each experiment was confirmed by AAN tretments (again in triplicate) of at least 1 strain in the presence of S9.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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

Method

Target gene:

For S. thyphimurium : histidine
For E. coli : tryptophan

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

For E. coli WP2, 2 tryptophan-requiring strains : WP2 pKM101 and WP2 uvrA pKM101.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial fraction

Test concentrations with justification for top dose:

An initial toxicity range-finder experiment was carried out in strain TA1535 only, using concentrations of nitric oxide up to a maximum exposure level of 200ppm ( a dose selected on the basis of previoulsy published data for nitric oxide in this test sytem). However, no toxic or mutagenic effects were observed. A second range-finder experiment was performed using the same tester strain, but using a range of doses up to a maximum of 5000ppm, in order to demonstrate the efficacy of the exposure systeme (by clearly showing either toxic or mutagenic effects). Whilst no toxic effects were noted, evidence of mutagenic activity was seen at exposure of 4000 and 5000 ppm, and the latter dose level was therefore retained as the maximum exposure level for both mutation experiments. No toxic effects were noted in any of the test strains in the mutation experiments (as would be indicated by a thinning of the background baterial lawn or marked reductions in revertant numbers).

Dose selected : 8; 40; 200; 1000; 5000 ppm for the first experiment.
50; 158; 500; 1580; 5000 ppm for the second experiment

Vehicle / solvent:

Nitrogen (carrier/ diluent gas)

Controls

Untreated negative controls:

yes

Remarks:

Carrier gas (nitrogen)

Negative solvent / vehicle controls:

yes

Remarks:

Carrier gas (nitrogen)

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

A constant flow exposure system was designed (with a gas flow rate of 40L/min) in order to minimise the build-up of other oxides of nitrogen within the exposure chamber (particularly nitrogen dioxide which is readily produced by the reaction of nitric oxide with oxygen). Nitrogen dioxide levels were monitored before and during exposure.

Evaluation criteria:

A test article was considered to be mutagenic if :
- the assay was valide (please see the acceptance criteria hereafter)
- Dunnett's test gave a significant response (p< or = to 0.01) and the data set showed a significant dose correlation
- The positive responses described in the previous section were reproductible

Acceptance criteria :
The assay was considered valid if the following criteria were met :
- The mean negative control counts fell within the normal ranges
- The positive control chemicals induces clear increases in revertant numbers confirming discrimination between different strains, and en active S-9 preparation
- No more than 5% of the plates were lost through contamination or some other unforeseen event.

Statistics:

Dunnett's test

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: S. typhimurium TA1535, TA 100, E. coli WP2 uvrA pkm101

Remarks:

Concentration > 1580 ppm

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: TA98; and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

It was concluded that nitric oxide clearly induced mutation in strains TA100 and TA1535 of Salmonella typhimurium. Additional evidence of this activity was also probably seen following some treatments of the Escherichia coli test strains. Mutagenic activity was observed both in the absence and presence of S9, but only at exposure concentration in excess of 1580 ppm.

Under tests conditions, test material exhibit a mutagenic activity on prokaryotic cells, with and without metabolic activation.