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Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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 / strainopen allclose all
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 / 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 resultsopen allclose all
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
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
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.