Dinitrogen oxide

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity of inhalation anaesthetics: trichloroethylene, divinyl ether, nitrous oxide and cyclopropane

Author:

Baden JM, Kelley M, Mazze RI, Simmon VF

Year:

1979

Bibliographic source:

Brit J Anaesth 51: 417–421 cited in 'Nitrous oxide [MAK Value Documentation, 1993]'

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dinitrogen oxide

EC Number:

233-032-0

EC Name:

Dinitrogen oxide

Cas Number:

10024-97-2

Molecular formula:

N2O

IUPAC Name:

nitrogen oxide

Test material form:

gas

Method

Target gene:

histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

Type of metabolic activation system:
- source of S9: Liver of arochlor induced male rats

Test concentrations with justification for top dose:

1, 3, 9, 27, 91 % (v/v)

Vehicle / solvent:

- Vehicle used: room air

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Positive control substance:

other: Vinylidene chloride (3%) for desiccator incubation 2-anthramine (2.5 µg/plate) for liquid experiments

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Desiccator incubation experiments
Bacteria-seeded glucose-minimal plates with or without S-9 mix were placed in 9-litre, air-tight desiccator jars. Gas volumes of nitrous oxide were added to the desiccators to give desired concentrations, which were verified at the beginning and end of exposure using an infra-red gas analyser.
The bacterial plates were exposed to the test compound in sealed desiccator jars for 8 h at 37 °C then the plates were removed from the desiccators and incubated for a further 40 h at 37 °C. Colonies on each plate were counted.
- Liquid incubation experiments
Bacterial cells, with or without S-9 mix, and various amounts of nitrous oxide were added to sterile, 15-mL stoppered tubes and rotated on a wheel for 2 h at 37 °C. The air phase concentrations for nitrous oxide in the air spaces above the reaction mixtures, were the same as those used in the desiccator experiments. Concentrations were measured at the beginning and end of exposure, using an infra-red gas analyser, and were found to vary by not more than 10% with time. Aliquots of the test sample were added to tubes of top agar, mixed and poured onto glucose-minimal medium plates. The plates were incubated for 2 days at 37 °C in air and colonies were counted.

Statistics:

Statistical analysis was by t test; P<0.05 was considered significant

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Nitrous oxide did not increase the number of revertants in strain TA100 when assayed either in desiccators or in liquid incubation. Similar negative results were obtained with strain TA1535. In contrast, plates exposed to the positive controls, vinylidene chloride or 2-anthramine showed 2.4-14.4-fold increases in the number of revertant colonies.