Sodium azide

Immunotoxicity

Administrative data

Endpoint:

immunotoxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

In vivo and in vitro effects of sodium azide on mouse complement

Author:

Johnson K.W. et al.

Year:

1984

Bibliographic source:

PMID: 6719470, Tox. Appl. Pharmacol. 73:559-63.

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: A microtiter hemolytic assay was utilized to determine sodium azide (NaN3) modulation of B6C3Fl and C3H mouse serum complement levels in vivo.
- Short description of test conditions: In three in vivo experiments, female B6C3Fl mice were exposed to NaN3 and physiological saline (vehicle control). Complement hemolytic ability was evaluated after a 1-day, single intravenous (iv) injection of 0.2, 2.0, and 20.0 mg/kg NaN3; at days 1, 2, 3, 4, and 6 of a 6 day time course study after intraperitoneal (ip) administration of 20 mg/kg NaN3; and at the end of an 11-day study involving daily injections of 10, 15, and 20 mg/kg NaN3 administered via ip.

GLP compliance:

no

Limit test:

no

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

Specific details on test material used for the study:

- Name of test material (as cited in study report): Sodium azide, NaN3
- Substance type: no data
- Physical state: no data
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: no data

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Litton (Frederick, MD, US)
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 17-20 grams
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 1 week quarantine

Administration / exposure

Route of administration:

other: 1- and 6-day study: intravenous; 11-day study: intraperitoneal

Vehicle:

physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Sodium azide was prepared weekly in physiol. saline (0.15 M NaCl).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

1-day, single injection
single injection with 6-day follow up
11-day, daily injections

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.2 other: mg/kg bw

Remarks:

1-day study

Doses / concentrations 2

Dose / conc.:

2 other: mg/kg bw

Remarks:

1-day study

Doses / concentrations 3

Dose / conc.:

10 other: mg/kg bw

Remarks:

11-day study

Doses / concentrations 4

Dose / conc.:

15 other: mg/kg bw

Remarks:

11-day study

Doses / concentrations 5

Dose / conc.:

20 other: mg/kg bw

Remarks:

1-day study; single injection with 6-day follow up study and 11-day study

No. of animals per sex per dose:

1-day, single injection and 6-day follow up: 6 animals (female) per group
11-day, daily injections: 8 animals (female) per group

Control animals:

yes, concurrent vehicle

Examinations

Other functional activity assays:

OTHER ASSAYS : Complement system determination
- Method: Based on Van Dijk's work (Van Dijk, 1980). However, the following modifications were made for a microtiter assay with a homologous system of mouse IgM and mouse complement. Following in vivo sodium azide treatment, blood was collected from chloroform-anesthetized mice by cardiac puncture. After allowing the blood to clot at room temperature for 2 h, serum was harvested and diluted 15 in ice cold Tris buffer (0.01 M Tris; 0.14 M NaCI; 0.15 mM CaCl2; 0.5 mM MgCl2; 0.1% gelatin; pH 7.3). Diluted serum was added in volumes of 20, 30, 50, 100, and 200 µL to 96 well V-bottom microtiter plates (Dynatech) and placed on ice. Each well contained 1.5 X 10^7 rabbit erythrocytes sensitized with a proper dilution of heat inactivated mouse anti-rabbit sera. Tris buffer was added to each well such that a total volume of 250 µL/well was obtained. After incubating at 30°C for 30 min, the plates were centrifuged at 650 g at 4°C for 10 min to pellet the erythrocytes and halt complement-mediated lysis.
From each well, 200 µL of supernatant fraction was placed in ABA-100 (bichromatic analyzer; Abbott Laboratories, North Chicago, Ill.) multicuvettes. The bichromatic analyzer measured amounts of hemoglobin present in two wavelengths (550 and 650 nm). The percentage of lysis was determined for each sample via comparison to a 100% water-lysed control. A Tris buffer blank containing opsonized erythrocytes and no serum was automatically subtracted from each reading to correct for spontaneous lysis. The ABA-100 data were analyzed by a program based on the Von Krogh equation (Mayer, 1961) which derives the CH50 value from the percentage lysis data for each sample. The functional complement activity was expressed in CH50 units per milliliter. The CH50 referred to the amount of complement lysing 50% of sensitized erythrocytes under standard conditions. Expressing functional complement activity in CH50 units was first proposed by Mayer ( 1961) and is the most acceptable method in both clinical and research settings.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Description (incidence):

For more details see "details on results" section below.

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

For more details see "details on results" section below.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not examined

Details on results:

As seen in Table 1, the effect of sodium azide on B6C3F1 complement haemolytic activity was limited.
The CH50 values (the amount of compartment lysing 50% of sensitized erythrocytes under standard conditions) for sodium azide-treated animals in the 1-day acute study were neither dose related nor significantly different from saline controls.
Animals exposed to 20 mg/kg sodium azide in the 6-day time course experiment displayed no significant changes in CH50 values. Of 36 treated animals, 3 died shortly after dosing during the 6-day study.
In the 11-day study, animals exposed to 10 mg/kg sodium azide exhibited significantly higher CH50 values. However, this effect was not considered biologically significant due to low control values and the lack of a dose-dependent relationship. Lethality also occurred among animals in the 11-day study. Two mice in the 15-mg/kg group and all animals in the 20-mg/kg group died of sodium azide-induced respiratory paralysis.

Specific immunotoxic examinations

Cell viabilities:

not examined

Humoral immunity examinations:

not examined

Specific cell-mediated immunity:

not examined

Non-specific cell-mediated immunity:

not examined

Other functional activity assays:

no effects observed

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

acute study (1-day)

Effect level:

<= 20 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: other functional activity assays (mouse complement mediated lysis of IgM sensitized rabbit erythrocytes)

2

Dose descriptor:

LOAEL

Remarks:

subacute study (11-day)

Effect level:

15 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Mortality

Any other information on results incl. tables

Table 1: The effect of sodium azide on B6C3F1 mouse complement levels following in vivo exposure

Experiment	Route	N	Exposure group	CH50 value^a
1-day, single injection	i.v.	6	Vehicle (NaCl)	107 ± 11.5
			0.2 mg/kg	144 ± 20.6
			2.0 mg/kg	98 ± 2.5
			20 mg/kg	114 ± 11.2
6-day time course after single dose (20 mg/kg)	i.v.	6	Vehicle	106 ± 6.6
			Day 1	136 ± 9.5
			Day 2	112 ± 9.6^b
			Day 3	102 ± 7.0
			Day 4	124 ± 15.9^c
			Day 6	117 ± 4.5
11-day, daily injection	i.p.	8	Vehicle	79 ± 2.0
			10 mg/kg	101 ± 7.5^d
			15 mg/kg	89 ± 9.9^e
			20 mg/kg	---^f

^aThe CH5O value reflects functional complement levels.

^bOne animal died (within 24 hr of dosing).

^cTwo animals died (within 24 hr of dosing).

^dValue differed significantly from control (p < 0.05).

^eTwo animals died by Day 11.

^fAll animals died by Day 4.

 

Applicant's summary and conclusion

Conclusions:

Sodium azide does not affect mouse complement levels in vivo.

Executive summary:

A microtiter hemolytic assay was utilized to determine sodium azide modulation of B6C3F1 and C3H mouse serum complement levels in vivo and in vitro. Functional complement was expressed in CH50 units per milliliter. Experiments were performed to determine the in vitro effect of Sodium azide on complement mediated lysis of IgM sensitized rabbit erythrocytes.

Concentrations of 5, 10, 20, 30, 40, 60, and 80 mM sodium azide were added to microtiter wells containing Tris buffer, IgM sensitized rabbit erythrocytes, and serum complement from naïve female C3H mice. Although NaCl and KCl controls had an inhibitory effect, Sodium azide demonstrated a significant dose-dependent inhibition of complement-mediated lysis.

In the three in vivo experiments, female B6C3FI mice were exposed to Sodium azide and physiological saline (vehicle control). Complement hemolytic ability was evaluated after a 1-day, single iv injection of 0.2, 2.0, and 20.0 mg/kg sodium azide; at Days 1, 2, 3, 4, and 6 of a 6-day time course study after ip administration of 20 mg/kg sodium azide; and at the end of an 11-day study involving daily injections of 10, 15, and 20 mg/kg sodium azide given ip.

No significant changes in complement-mediated hemolysis were observed in the in vivo experiments. These studies indicate that Sodium azide does not affect mouse complement levels in vivo.