Sodium azide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Effect of sodium azide on sister-chromatid exchanges in human lymphocytes and Chinese hamster cells

Author:

Arenaz P., Nilan R.A.

Year:

1981

Bibliographic source:

Mutation Research 88, 217-221

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

: no metabolic activation, highest concentration shows no clear sign of toxicity

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

Sodium azide

EC Number:

247-852-1

EC Name:

Sodium azide

Cas Number:

26628-22-8

Molecular formula:

N3Na

IUPAC Name:

sodium azide

Method

Target gene:

no data

Species / strain

Species / strain 1

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Mc Coys 5A medium + 15% FCS + 0.1% gentamycin

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Gibco chromosome medium 1A, supplemented with phytohemagglutin

Metabolic activation:

without

Test concentrations with justification for top dose:

lymphocytes: 1h exposure: 0.5 mM - 50 mM mol/L
lymphocytes: 4h exposure: 0.1 µM - 100 µM mol/L
CHO Cells: 2h exposure: 0.5 µM - 1 mM mol/L

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: physiol. saline, pH 6.8

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.05 µg/mL

Details on test system and experimental conditions:

CHO:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): ~5 X10^5

DURATION
- Preincubation period: 24 hours
- Exposure duration: 2 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Deduced 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa (Perry and Wolff 1974)

NUMBER OF REPLICATIONS: One

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not specified except by reference (Perry and Wolff 1974)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 49 - 51

DETERMINATION OF CYTOTOXICITY
- Method: Only indicated cultures with lethality

Human lymphocytes:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 1 or 4 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Deduced 48 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa (Perry and Wolff 1974)

NUMBER OF REPLICATIONS: 2 - 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not specified except by reference (Perry and Wolff 1974)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 40 - 120 (number of cells evaluated for all the replication tests)

Evaluation criteria:

no data

Statistics:

not specified

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Lethal at 5 X 10^-5 M and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

slightly but significantly increased. However, the increase was probably due to the inhibition by azide of catalase and peroxidase which would lead to an increase in H2O2, a known inducer of SCEs.

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

CHO cells:
- No increase in SCE after 2h exposure of CHO cells with concentrations from 0.5 µM - 10 µM.
- Cytotoxicity observed in CHO cells at concentrations exceeding 50 µM.

Lymphocytes:
- Slight but significant increase of SCE at 1h exposure to 50 mM.
- No increase in SCE of human lymphocytes after 4h exposure up to 100 µM.

Remarks on result:

other: 2h treatment

Any other information on results incl. tables

Table 1: Effects of various concentrations of sodium azide on SCE induction in human lymphocytes

Treatment dose		Treatment time [h]	Number of cells analyzed	SCEs/cell ± SE
100 µM	a	4	80	8.00 ±0.307
50 µM	b	4	117	7.25 ±0.241
10 µM	b	4	110	7.65 ±0.297
5 µM	b	4	91	7.41 ±0.260
1 µM	b	4	120	7.79 ±0.239
0.5 µM	a	4	80	7.48 ±0.334
0.1 µM	a	4	80	6.88 ±0.261
0.05 µg/mL Mitomycin C	b	4	92	45.84 ±1.23 *
Control			120	7.48 ±0.244
50 mM	a	1	40	10.35 ±0.647 *
10 mM	a	1	64	9.03 ±0.549
5 mM	a	1	67	9.40 ±0.471 *
1 mM		1	20	8.63 ±0.467
0.5 mM		1	40	8.03 ±0.492
0.05 µg/mL Mitomycin C	a	4	40	38.03 ±1.80 *
Control			60	8.113 ±0.326

a Average of 2 experiments

b Average of 3 experiments

* Significant (p<0.05)

 

Table 2: Effects of various concentrations of sodium azide on SCE induction inChinese hamster K1 cell line

Treatment dose		Treatment time [h]	Number of cells analyzed	SCEs/cell ± SE
1 mM		2	---	Lethal
500 µM		2	---	Lethal
100 µM		2	---	Lethal
50 µM		2	---	Lethal
10 µM		2	50	11.20 ±0.512
5 µM		2	49	11.90 ±0.456
1 µM		2	51	12.05 ±0.490
0.5 µM		2	50	10.46 ±0.554
Control		2	50	10.70 ±0.572

 

 

Applicant's summary and conclusion

Conclusions:

The data suggest, that sodium azide does not interact with DNA in a manner that either produces SCEs in CHO cells. A slight but significant increase of SCEs in human lymphocytes has been observed after 1h incubation with 50 mM sodium azide. However, this increase was probably due to the inhibition by azide of catalase and peroxidase, which would lead to an increase in H2O2, a known inducer of SCEs.

Executive summary:

Previous reports from this laboratory and others indicate that sodium azide is a unique mutagen. It is highly mutagenic in S. typhimurium TA1530 as well as in barley, rice, peas, and yeast. However, azide apparently does not produce chromosome breaks in barley, vicia or human lymphocytes.

In a mammalian cell cytogenetics assay (Sister-chromatid exchanges, SCE), human whole blood or Chinese hamster K1 (CHO) cells were exposed for 1, 2, or 4 h respectively to various concentrations of sodium azide ranging from 10^-7 to 5 x 10^-2 M in the absence of metablic activation. Cells were harvested and chromosomes stained by the FPG technique. In human lymphocytes, concentrations above 10^-4 induced lethality (after 4h treatment) whereas CHO cells were sensitive to concentrations up to 10^-5M. The lower concentrations of azide produced no significant increase in SCE frequency above controls; only in the highest concentration of the 1h treatment group (50 mM) a slight increase of SCE could be seen. Concurrent mitomycin C treatments produced significant increases in SCE levels. The presented data suggest, that this highly mutagenic compound does not interact with DNA in a manner that either produces chromosome aberrations or SCEs. Thus, azide appears to be unique among mutagenic compounds. In fact, it has been suggested that azide acts strictly as a base substitution mutagen. It has been reported that azide requires metabolic activation in both barley and S. typhimurium. To date, however no mutagenic metabolite has been detected in mammalian cells.