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.
POTASSIUM NITRITE
CHEMICAL NAME Potassium nitrite
EMPIRICAL FORMULA KNO2
MOLECULAR WEIGHT 85.11
DEFINITION Potassium nitrite after drying contains not less
than 95% of KNO2. It conforms to the following
specifications.
DESCRIPTION Small, white or slightly yellow, deliquescent
granules or rods.
USE As a colour fixative in fish products and in
pickling and curing meat, sometimes in
combination with sodium nitrite and with
potassium and sodium nitrates.
IDENTIFICATION TESTS
A. Solubility: Water: Very soluble
Ethanol: Sparingly soluble
B. In neutral, concentrated or moderately concentrated solutions of
potassium nitrite, sodium bitartrate TS slowly produces a white,
crystalline precipitate which is soluble in ammonia TS and in
solutions of alkali hydroxide or carbonates.
C. Potassium nitrite yields brownish red fumes when treated with
diluted mineral acids or acetic acids.
PURITY TESTS
Arsenic: Not more than 3 mg/kg.
Lead: Not more than 10 mg/kg.
Heavy metals: Not more than 20 mg/kg.
Dissolve 2 g in water, and dilute to 40 ml. For the control, add a
volume of standard lead TS equivalent to 0.02 mg of lead to 10 ml of
this solution and use the remaining 30 ml for the sample. Evaporate
both solutions to dryness on the steam bath. To each residue add 5 ml
of hydrochloric acid, and again evaporate to dryness. Dissolve the
residues in 20-ml portions of water, adjust each to a pH between 3.0
and 4.0, dilute to 40 ml and add 10 ml of hydrogen sulfide TS to each.
The colour in the sample solution, measured in a 50-ml Nessler tube,
is not darker than the control solution, similarly measured.
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 6.0 and 9.0.
ASSAY
Dissolve 1.000 g, previously dried over silica gel for 4 hours, in
water and make to 100.0 ml. Pipette 10 ml of this solution into a
mixture of 50.0 ml of 0.1 N potassium permanganate, 100 ml of water
and 5 ml of sulfuric acid, keeping the tip of the pipette well below
the surface of the liquid. Warm the solution to 40°C, allow it to
stand for 5 minutes and add 25.0 ml of 0.1 N oxalic acid. Heat the
mixture to about 80°C and titrate with 0.1 N potassium permanganate.
Each ml of 0.1 N potassium permanganate is equivalent to 4.256 mg of
KNO2.
SODIUM NITRITE
CHEMICAL NAME Sodium nitrite
EMPIRICAL FORMULA NaNO2
MOLECULAR WEIGHT 69.00
DEFINITION Sodium nitrite after drying contains not less
than 97% of NaNO2. It conforms to the
following specifications.
DESCRIPTION White or slightly yellow, hygroscopic granules,
powder, or opaque, fused masses of sticks. It
is deliquescent in air.
USE As a colour fixative in fish products and in
pickling and curing meat, sometimes in
combination with potassium nitrite, or
potassium and sodium nitrates.
IDENTIFICATION TESTS
A. Solubility: Water: 1 g dissolves in 1.5 ml
Ethanol: Sparingly soluble
B. Sodium nitrite, after conversion to chloride or nitrate, yields
with uranyl zinc acetate TS a golden-yellow precipitate, which
forms after several minutes' agitation.
C. Sodium nitrite yields brownish red fumes when treated with
diluted mineral acids or acetic acid.
PURITY TESTS
Loss on drying: Not more than 0.25% by drying over silica gel for 4
hours.
pH of a 5% solution: Dissolve 10g in 200 ml of carbon dioxide- and
ammonia-free waters: the pH of this solution is between 6.0 and 9.0.
Arsenic: Not more than 3 mg/kg.
Lead: Not more than 10 mg/kg.
Heavy metals: Not more than 20 mg/kg.
Dissolve 2 g in water and dilute to 40 ml. For the control, add a
volume of standard lead solution equivalent to 0.02 mg of lead to 10
ml of this solution and use the remaining 30 ml for the sample.
Evaporate both solutions to dryness on the steam bath. To each
residue add 5 ml of hydrochloric acid, and again evaporate to dryness.
Dissolve the residues in 20-ml portions of water, adjust each to a pH
between 3.0 and 4.0, dilute to 40 ml and add 10 ml of hydrogen sulfide
TS to each. The colour in the sample solution, measured in a 50-ml
Nessler tube, is not darker than the control solution, similarly
measured.
ASSAY
Dissolve 1.000 g, previously dried over silica gel for 4 hours, in
water and make to 100.0 ml. Pipette 10 ml of this solution into a
mixture of 50.0 ml of 0.1 N potassium permanganate, 100 ml of water
and 5 Ml of sulfuric acid, keeping the tip of the pipette well below
the surface of the liquid. Warm the solution to 40°C, allow it to
stand for 5 minutes, and add 25.0 ml of 0.1 N oxalic acid. Heat the
mixture to about 80°C and titrate with 0.1 N potassium permanganate.
Each ml of 0.1 N potassium permanganate is equivalent to 3.450 mg of
NaNO2.
Biological Data
Biochemical Aspects
Following absorption of nitrites, the most important biochemical
reaction that occurs is the conversion of haemoglobin to
methaemoglobin. There is some controversy concerning the molar ratios
involved in this reaction. Making an extreme assumption, it may be
stated that 1 g of sodium nitrite could convert as much as 1855 g of
haemoglobin to methaemoglobin.1
The sub-acute hazard of nitrites rests on the amount of methaemoglobin
formed and on the ability of the body to reconvert this methaemoglobin
back to haemoglobin.
Acute toxicity
Animal Route LD50 Reference
(mg/kg body-weight)
Mouse oral 220 2
Mouse-female oral 175 3
Rat-female oral 85 3
Many cases have been reported of accidental poisoning resulting from
the presence of sodium nitrite in food products. From this
information it is possible to deduce that the oral lethal dose in man
varies from 0.18 to 2.5 g, the lower figures being those for children
and old people.4,5,6,7 Sodium nitrite has been used for therapeutic
purposes as a vasodilating agent in dosages of 30-120 mg.
Short-term studies
Rat and cat. Rats were fed a sodium nitrite supplement for a period
up to 168 days. One rat received a total of 167 mg of sodium nitrite
in 121 days. This represents 93 ppm in the daily diet. No effects on
growth or on the weights of important organs were noted. In a similar
experiment with cats, one animal received a total of about 4100 mg of
sodium nitrite during a period of 105 days. This represents
approximately 390 ppm in the daily diet. No effects on the growth
rate or on the weight of important organs were noted. No
histopathological examination has been reported on any animal fed with
nitrite.
Long-term studies
Rat. The continuous administration of sodium nitrite in the
drinking-water at the rate of 100 mg/kg body-weight daily over the
whole life span and in 3 successive generations (95 rats) resulted in
spite of the high dosage (67% of the acute LD50) in only a slight
inhibition of growth (10-20%) and in a shortening of the median life
span from 740 to 640 days. Reproduction was normal. Neither the
blood picture nor the organs showed any ill effects. The number of
tumours observed in the test group (1 thymoma and 1 hepatoma) was not
greater than in the control group. Cumulative toxic effects were not
observed.9
Since nitrous acid reacts easily with secondary amines yielding
dialkylnitrosamines which are highly toxic and produce cancer of the
liver in rats, additional experiments have been made in which nitrite
was administered together with diethylamine. Thirty hybrid rats were
given sodium nitrite in the drinking-water at a daily dosage of 100
mg/kg body-weight and diethylamine in the food at the rate of
500 mg/kg body-weight daily. The same slight shortening of the median
life span (to 625 days) was observed as in the experiments with
nitrite alone, but there were no tumours.9
Comments on experimental studies reported
Though in the long-term studies cited only a slight inhibition of
growth occurred, the dose causing this effect appears to give the best
approximation to the threshold dose level.
Evaluation
Level causing no significant toxicological effect in the rat
From consideration of the long-term studies it can be concluded that
this level will be somewhat below 100 mg/kg body-weight per day.
Estimate of acceptable daily intakes for man
mg/kg body-weight
Unconditional acceptance 0-0.4
Conditional acceptance 0.4-0.8
Comment
It is recommended that if possible sodium nitrite should be used mixed
with common salt, the amount of nitrite in the salt being not more
than 0.6%. Food for babies should not contain added nitrite.
Further Work Considered Desirable
1. The estimation of nitrite in vegetables.
2. Chronic toxicity experiments in non-rodent species.
References
1. Lehman, A. J. (1958) Quart. Bull. Ass. Food Drug. Off., 22, 136
2. Riemann, H. (1950) Acta pharmacol. (Kbh.), 6, 285
3. United States Food and Drug Administration (Unpublished data)
4. Schrader, G. & Gessner, O. (1943) Samml. Vergiftungsf., 13, 101
5. Greenberg, M., Birnkrant, W. B. & Schiftner, J. J. (1945) Amer. J.
publ. Hlth., 35, 1217
6. Schmidt, H., Stick, W. & Kluge, F. (1949) Dtsch. med. Wschr., 74,
961
7. Naidu, S. R. & Venkratrao, P. (1945) Calcutta med. J., 42, 79
8. Tarr, H. L. A. & Carter, M. H. (1942) J. Fish. Res. Board, Can.,
6, 63
9. Druckrey, H. (1963) Arzneimittel-Forsch., 13, 320