WHO/Food Add./24.65
FAO Nutrition Meetings
Report Series No. 38A
SPECIFICATIONS FOR IDENTITY AND
PURITY AND TOXICOLOGICAL EVALUATION
OF SOME ANTIMICROBIALS AND
ANTIOXIDANTS
The content of this document is the result of the deliberations of the
Joint FAO/WHO Expert Committee on Food Additives which met 8-17
December 1964a
a Eighth Report of the Joint FAO/WHO Expert Committee on Food
Additives, Wld Hlth Org. techn. Rep. Ser., 1965, 309; FAO
Nutrition Meetings Report Series 1965, 38.
SODIUM NITRATE
CHEMICAL NAME Sodium nitrate
SYNONYMS Chile saltpetre; cubic or soda nitre
EMPIRICAL FORMULA NaNO3
MOLECULAR WEIGHT 85.00
DEFINITION Sodium nitrate after drying contains not less
than 99.0% of NaNO3 and conforms to the
following specifications.
DESCRIPTION Clear, colourless, odourless, transparent
crystals, or white granules or powder.
Deliquescent in moist air.
USE As an antimicrobial preservative and colour
fixative in meat, fish and cheese products.
Often need In combination with nitrites.
IDENTIFICATION TESTS
A. Solubility: Water: 1 g is soluble in 1.1 ml
Ethanol: 1 g is soluble in 125 ml
Glycerol: Slightly soluble
B. Sodium nitrate yields with uranyl zinc acetate TS a golden-yellow
precipitate, which forms after several minutes' agitation.
C. Brownish red fumes are evolved when sodium nitrate is heated with
sulfuric acid and metallic copper.
PURITY TESTS
Loss on drying: Not more than 2% after drying at 105°C for 4 hours.
pH of a 5% solution: Dissolve 10 g in 200 ml of carbon dioxide- and
ammonia-water, the pH of this solution is between 5.5 and 8.3.
Arsenic: Not more than 3 mg/kg.
Lead: Not more than 10 mg/kg.
Nitrite: Not more than 1000 mg/kg.
Dissolve 1 g in 50 ml of carbon dioxide- and ammonia-free water, add 2
drops of dilute ammonia TS, and filter if necessary through paper into
a 100-ml volumetric fleck. Add 10 ml of dilute acetic acid TS, shake
the mixture, and allow to stand for 5 minutes. Any colour produced is
not darker than that of a complete blank to which 1 ml of nitrite
standard TS is added.
Heavy metals: Not more than 20 mg/kg.
Dissolve 3 g in water and dilute to 30 ml. Adjust 20 ml of this
solution to a pH between 3.0 and 4.0 and dilute with water to 40 ml.
To the remaining 10 ml of sample solution add water to make 35 ml, add
a volume of standard lead TS equivalent to 0.02 mg of lead and adjust
to a pH between 3.0 and 4.0 and dilute to 40 ml. To each solution add
10 ml of hydrogen sulfide TS, mix, transfer to 50-ml Nessler tubes and
observe after 10 minutes. The colour of the test solution should be
no darker than that of the solution containing the standard lead
solution.
ASSAY
Weigh 1.000 g of sodium nitrate previously dried at 105°C for 4 hours
into the flask of an ammonia distillation apparatus. Add 10 g of
Devarda's Alloya and 25 ml of N sulfuric acid and allow the flask
to stand, protected from ammonia in the atmosphere, for 1 hour. Add
100 ml of a solution of sodium hydroxide (15 in 100), connect the
flask to the remainder of the apparatus and distil 100 ml, collecting
the distillate under the surface of 25.0 ml of N hydrochloric acid.
Rinse the condenser into the receiving flask, add methyl red-methylene
blue TS and titrate the excess acid with N sodium hydroxide.
(25-x)
%NaNO3 = × 8.5
w
POTASSIUM NITRATE
CHEMICAL NAME Potassium nitrate
SYNONYMS Saltpetre, nitre
EMPIRICAL FORMULA KNO3
MOLECULAR WEIGHT 101.11
DEFINITION Potassium nitrate after drying contains not
less than 99% of KNO3 and conforms to the
following specifications.
DESCRIPTION Colourless, odourless, transparent prisms, or
white granular or crystalline powder, having a
cooling, saline, pungent taste.
a Devarda's Alloy (Devarda's Metal) is a grey powder composed of 50
parts of copper, 45 parts of aluminium and 5 parts of zinc.
USE As an antimicrobial preservative and colour
fixative in meat, fish and cheese products;
often used in combination with nitrites.
IDENTIFICATION TESTS
A. Solubility: Water: 1 g is soluble in 3 ml
Ethanol: 1 g is soluble In 620 ml
Glycerol: Soluble
B. In neutral, concentrated or moderately concentrated solutions of
potassium salts, sodium bitartrate TS slowly produces a white,
crystalline precipitate which is soluble in ammonia TS and in
solutions of alkali hydroxides or carbonates.
C. Brownish red fumes are evolved when potassium nitrate is heated
with sulfuric acid and metallic copper.
PURITY TESTS
Loss on drying: Dry at 105°C for 4 hours: it loses not more than 1%
of its weight.
pH of a 5% solution: Dissolve 10 g in 200 ml of carbon dioxide- and
ammonia-free water: the pH of this solution is between 4.5 and 8.5.
Arsenic: Not more than 3 mg/kg.
Lead: Not more than 5 mg/kg.
Heavy metals: Not more than 20 mg/kg.
Dissolve 3 g in water and dilute to 30 ml. Adjust 20 ml of this
solution to a pH between 3.0 and 4.0 and dilute with water to 40 ml.
To the remaining 10 ml of sample solution add water to make 35 ml, add
a volume of standard lead TS equivalent to 0.02 mg of lead, and adjust
to a pH between 3.0 and 4.0, and dilute to 40 ml. To each solution
add 10 ml of hydrogen sulfide TS, mix, transfer to 50-ml Nessler
tubes and observe after 10 minutes. The colour of the test solution
should be no darker than that of the solution containing the standard
lead solution.
Nitrite: Not more than 1000 mg/kg.
Dissolve 1 g in 50 ml of carbon dioxide- and ammonia-free water, add 2
drops of diluted ammonia TS, and filter if necessary through paper
into a 100-ml volumetric flask. Add 10 ml of potassium iodide TS,
2 ml of starch TS, and 10 ml of diluted acetic acid TS, shake the
mixture and allow to stand for 5 minutes. Any colour produced is not
darker than that of a complete blank to which 1 ml of nitrite standard
TS is added.
ASSAY
Weigh 1.000 g of potassium nitrate previously dried at 105°C for 4
hours into the flask of an ammonia distillation apparatus. Add 10 g
of Devarda's Alloy and 25 ml of 1 N sulfuric acid and allow the
flask to stand, protected from ammonia in the atmosphere, for 1 hour.
Add 100 ml of a solution of sodium hydroxide (15 in 100), connect the
flask to the reminder of the apparatus and distil 100 ml, collecting
the distillate under the surface of 25.0 ml of 1 N hydrochloric
acid. Rinse the condenser into the receiving flask, add methyl
red-methylene blue TS and titrate the excess acid with 1 N sodium
hydroxide.
(25-x)
%KNO3 = × 10.11
w
Biological Data
Biochemical aspects
In certain circumstances reduction of nitrate to nitrite can take
place in the digestive tract by the activity of the intestinal flora.
If appreciable reduction occurs before the normal rapid elimination of
the nitrate, poisoning can result. This appears to have occurred in
cattle,1 and in babies less than 6 months old, especially in
dyspeptic infants.2
In experiments with rabbits about one-half of the ingested amount of
nitrate was excreted in the urine, and only 0.5% was recovered as
nitrite in the urine.3
Acute toxicity
Animal Route Minimum lethal dose LD50 Reference
(mg/kg body-weight) (mg/kg
body-weight)
Rat-male oral 190-2000 - 4
Rat-female oral 460-1200 - 4
Rat oral - 3236 5
Poisoning in man may result from a total oral daily dose in excess of
4 g or from a single dose of more than 1 g. 8 g may be fatal and
13-15 g are generally fatal.6
Short-term studies
Cattle. Most data have been obtained from livestock fed various
forage crops with a high nitrate content. Poisoning depends upon the
conversion of the nitrate to nitrite by the intestinal flora. The
lowest level that may result in fatal poisoning in cattle has been
reported to be 1.5% of potassium nitrate in the forage.1
Dog. Two dogs were fed 2% of sodium nitrate in their diets for 105
days and for 125 days without any adverse effects.7
Man. Numerous cases have been reported of poisoning in small
children and infants from the use of well-water containing nitrates.
Among these, them were 26 cases in which the nitrate nitrogen content
of the well-water was 21-50 ppm (93-221 ppm as NO3), 54 cases in
which it was 51-100 ppm (221-443 ppm as NO3), and 52 cases in which
it was over 100 ppm (443 ppm as NO3).
In one instance, a level as low as 50 ppm (as NO3) in tap water
produced 72% methaemoglobinaemia in a dyspeptic child.2 Healthy
babies, however, have tolerated quantities up to 21 mg/kg body-weight
(as NO3) for one week without any disturbance.9
Long-term studies
Rat. Sodium nitrate was fed to 4 groups of 20 rats each at dosages of
0.1%, 1%, 5% and 10% of the diet for 2 years. Slight growth
depression occurred at the 5% level, and additional morphological
changes due to inanition occurred at the 10% level.7
Evaluation
Levels causing no significant toxicological effect in animals
From consideration of the long-term study in rats, the level of sodium
nitrate causing no demonstrable effect over a period approximating to
the life span is assessed at 1% of the diet, or 500 mg/kg body-weight
daily. In the short-term study with dogs fed 2% sodium nitrate in the
diet for 105 days, the level producing no demonstrable effect likewise
corresponds to 500 mg/kg body-weight per day.
Estimate of acceptable daily intakes for man
mg/kg body-weight
(as sodium nitrate)
Unconditional acceptance 0-5
Conditional acceptance 5-10
Comment
In establishing tolerances for added nitrate it is important to take
into account the amount of nitrate already present in other foods.
The sensitivity of normal babies and the apparent sensitivity of
dyspeptic babies makes it impossible to make an estimate of an
acceptable dose for babies of 6 months of age or less on the basis of
animal experimentation or clinical experience. Nitrate should on no
account be added to baby-foods. Water with high nitrate content is
unsuitable for the preparation of baby-foods.
It is recommended that, if possible, sodium nitrate should be used as
a 2.5% mixture with common salt.
References
1. Bradley, W. B., Eppson, H. F. & Beath, O. A. (1940) Wyom. Univ.
agr. exp. Stat. Bull., 24l, 20
2. Thal, W., Lachhein, L. & Martinek, M. (1961) Arch. Toxikol., 19,
25
3. Kilgore, L., Almon, L. & Geiger, M. (1959) J. Nutr., 69, 39
4. Spector, W. S. ed. (1956) Handbook of toxicology, Saunders,
Philadelphia & London, vol. 1
5. United States Food and Drug Administration (Unpublished data)
6. Sollmann, T. (1957) A manual of pharmacology, Saunders,
Philadelphia & London
7. Lehman, A. J. (1958) Quart. Bull. Ass. Food Drug Off., 22, 136
8. Rosenfield, A. B. & Huston, R. (1950) Minn. Med., 33, 787
9. Kübler, W. (1958) Z. Kinderheilk., 81, 405