420. Glycerol and glycerol diacetate (WHO Food Additives Series 10)

TOXICOLOGICAL EVALUATION OF CERTAIN FOOD ADDITIVES

WHO FOOD ADDITIVES SERIES 10

The evaluations contained in this document were prepared by the
Joint FAO/WHO Expert Committee on Food Additives*
Rome, 21-29 April 1976

Food and Agriculture Organization of the United Nations

World Health Organization

*Twentieth Report of the Joint FAO/WHO Expert Committee on Food
Additives, Geneva, 1976, WHO Technical Report Series No. 599, FAO Food
and Nutrition Series No. 1.

Glycerol and glycerol di-acetate

(Food Grade)

Explanation

Glycerol occurs naturally in varied combined forms as a simple,
mixed or complex glyceride in association with fatty acids,
carbohydrates, phosphate or amino acid. It rarely exists in
significant amounts in the free form in natural fats or oils of
animal, vegetable, marine or biological origin. Glycerol constitutes
about 10% of the lipid molecule. Natural glycerol is isolated as a
by-product from the hydrolysis of fats and oils during the manufacture
of soaps, etc. Trace contaminants include fatty acids, their esters,
and processing chemicals including background levels of heavy metals.

Synthetic glycerol is produced by the hydrogenolysis of
carbohydrate material (cane or corn syrup sugar), or from propylene
and chlorine by a number of different processes including
chlorination, chlorohydrination and hydrolysis. Natural and synthetic
glycerine contain trace quantities of low-boiling components. Specific
analysis of other components of one synthetic glycerine prepared from
the hydrogenolysis of chlorhydrate material showed that it contained
no more than 0.1%, 1,2,3-butanetriol and 0.1% of 1,2,4-butanetriol.
The 1,2,3-butanetriol consists of erythro and threo isomers in the
approximate proportion of 90% of the former to 10% of the latter
(Atlas Chemical Ind., Inc., 1961).

Glycerol prepared from propylene was shown to be free of aromatic
compounds. The only specific contaminants identified were glycerine
polymers and glyceraldehyde (Hine et al., 1953).

Biological Data

Biochemical Aspects

Glycerol is readily metabolized and deposited as glycogen in
the liver of young white rats (Catron & Lewis, 1929). Glycerol is
metabolized by rats in a similar manner when administered
intraperitoneally, intragastrically or intravenously. ¹⁴C-Glycerol
administration results in a net synthesis of blood glucose and liver
glycogen, as well as resulting in ¹⁴C incorporation into the lipids
(neutral fat and phosphatides) of most tissues (Gidez & Karnovsky,
1954). Administration of glycerol to 5 to 10 day-old rats resulted in
a hepatic glycogenesis. This response was diminished in weaned and
almost absent in animals less than 48 hrs old (Johnson et al., 1971).

Phlorizinized dogs administered glycerol via stomach tube
converted it almost completely to glucose which was excreted in the
urine (Chambers & Deuel, 1925).

Analogy with triacetin and other di and triglycerides of fatty
acids shows that glycerol di-acetates will be rapidly hydrolysed by
hydrolytic enzymes excreted into the gastrointestinal tract as well as
by esterases present in all tissues. The resulting acetate and
glycerol will be rapidly and completely metabolized.

TOXICOLOGICAL STUDIES

Special studies on reproduction

Rat

Groups of 9 males and 18 females were maintained on synthetic
diets containing 30% glycerol. Rats receiving this diet were carried
through seven successive generations. Young produced from females on
the diet weighed on average 20% less than those from control females.
Studies utilizing lower levels of glycerol were not successful because
of nutritional inadequacies of the synthetic diet (Guerrant et al.,
1947).

In another study rats were maintained on diets containing 61%
starch and no glycerol, or 20% starch plus 41% glycerol, or no starch
plus 61% glycerol. Reproduction was not impaired by diets containing
41% glycerol. However, no pregnancies occurred on diets containing 61%
glycerol, presumably due to the inadequate nutritional quality of the
diet (Johnson et al., 1933).

Groups each of 20 rats (equally divided by sex) were dosed daily
per os with either 1.0 ml distilled water or 1.0 ml of a 20% aqueous
solution of glycerol/100 g body wt for eight weeks. Animals (P) from
each group were then bred. The pregnant rats in each group were then
divided into two groups of 5, one group continued to be dosed during
pregnancy, and for 12 weeks subsequent to giving birth to the pups.
The growth of the pups (F1) was monitored for 60 days. At 100 days
some of the F1 generation were killed for histological examination of
the reproductive organs. Ten males and ten females of the F1
generation were mated on maturity. However, these animals were not
administered glycerol. The F2 generation was monitored for growth and
onset of estrus cycle. Administration of glycerol to the P rats had no
effect on reproductive performance or growth of offsprings and onset
of estrus cycle of F1 and F2 generations (Wegener, 1953).

Acute toxicity

(a) Glycerol di-acetate

LD₅₀ Reference
Substance Species Route mg/kg body weight

Glycerol Mouse Oral 8.5 ml/kg BW Spector, 1956

Diacetate Mouse s.c. 2.5 ml/kg BW "

Mouse i.v. 2.3 ml/kg BW "

Rat s.c. 4.0 ml/kg BW "

(b) Glycerol

Oral LD50 mg/kg
Animal Origin of glycerol Reference
Natural Synthetic 1 Synthetic 2

Rat 27.5 - - Smyth et al., 1941

Rat 27.2 27.2 - Hine et al.,
(female) 1953

Rat 17.2 - 22.7 - 19.4 - 26.6 Atlas Co.,
(female) 1961

Rat 21.1 - 28.8 - 19.8 - 26.4 Atlas Co.,
(male) 1961

Mice 20.65 ± 0.47^a 20.81 ± 0.58^a - Anderson et al., 1950

Mice 23 ± 1.3 23 ± 1.9 - Hine et al.,
(male) 1953

Guinea pig 7.75 - - Smyth et al., 1941

Guinea pig 10 ± 1.3 11.5 ± 2.8 - Hine et al.,
(male) 1953

Rabbit 14 - 18^a - - Deichmann et al,, 1941

^a cc/kg.
Synthetic 1 - Prepared from propylene.
Synthetic 2 - Prepared from carbohydrate material.

Short-term Studies

Rat

Groups each of 5 female rats were given tap water to drink
containing 0, or 5% by volume natural glycerin, or 5% by volume
synthetic glycerin (prepared from propylene) for a period of 16 weeks.
Ground rat rations were allowed ad lib. Hematologic studies
(hemoglobin, erythrocytes, leucocytes and differentials) were made at
monthly intervals. At the end of the test period the animals were
sacrificed and submitted to necropsy. Gross and microscopic studies
were made of the heart, lungs, spleen, stomach, intestines, kidneys,
thymus, thyroid and adrenals. Weight gain was slightly increased in
groups receiving glycerol. Hematologic parameters in test and control
animals were similar. Histopathologic studies indicated that rats
(3/5) in the groups receiving glycerol (both natural and synthetic)
showed that several tubules near the junction of the cortex and
medulla contained calcified masses. No other compound related effects
were observed (Anderson et al., 1959).

In another study groups each of 20 rats were administered daily
per os either water, or 10 ml/kg body wt glycerol or at 20% aqueous
solution. Dosing was for a period of 40 days. At the end of the test
period the animals were autopsied, and histological examination made
of the liver, kidneys, bladder and small intestine. The only compound
related effect reported was calcium deposits in the urinary canals
(Kopf et al., 1951).

Groups each of 18 rats (Sprague Dawley strain), equally divided
by sex, were maintained on diets containing 0, or 20%, 10% or 5%
natural glycerol (99.8% glycerol, 0.02% low boilers) or 20%, 10% or 5%
synthetic glycerol prepared by the hydrogenolysis of carbohydrate
products (99.76% glycerol, 0.04% low boilers; and 1,2,3-Butanetriol
0.11%, and 1,2,4-Butanetriol 0.09%) for 50 weeks. The protein in the
test diets was balanced with that in the control diet by incorporation
of casein. The calorie density of all diets was similar. There were no
significant differences in the growth rate of the test and control
animals. Gross pathological examination of tissues and organs showed
that the only possible compound related effect was pituitary growths
which accounted for the largest segment of all of the observed
growths. However, there was no pattern or difference with respect to
the source of glycerol for any of these growths. Microscopic
examination of the tissues and organs showed no significant pathology
that could be related to dose or type of glycerin. Frequent changes
were associated with the kidney (tubular damage, protein counts,
inflammation and glomerular fibrosis). In male rats there was no
significant difference in the incidence between test and control. In
the female, no pathological findings were reported in the control
animals, but the incidence in the test groups was similar to that
observed in the test males (Atlas Chemical Co., 1969).

Long-term Studies

Rat

Groups each of 48 rats (Sprague Dawley strain) equally divided by
sex were maintained on diets containing 0, 20%, 10% or 5% natural
glycerol (99.8% glycerol, 0.02% low boilers), or 20%, 10% or 5%
synthetic glycerol prepared by the hydrogenolysis of carbohydrate
products (99.76% glycerol, 0.04% low boilers, and 1,2,3-Butanetriol
and 1,2,4-Butanetriol, 0.09%) for two years. The protein in the test
diets was balanced with that in the control by incorporation of
casein. The caloric density of the test and control diets was similar.
The following parameters were measured during the study: mortality,
growth, efficiency of caloric utilization, water consumption, volume
of urine excreted, urinalysis (including specific gravity, sediment,
acetone, albumin, sugar and oxalate), hematologic studies (including
white blood cells and differential count, red blood cells, hematocrit
and hemoglobin) and biochemical studies (including blood glucose, urea
nitrogen, plasma cholesterol, serum alkaline phosphatase and
transaminases), and bromsulfalein retention. At the termination of the
study the animals were killed, and subjected to a complete autopsy,
including determination of organ/body weight ratios, as well as gross
and microscopic examination of the tissues and organs. Rats fed diets
containing glycerol gained weight more rapidly than control rats.
Urine analysis, hematologic and biochemical determinations were
similar for test and control animals. Male rats fed diets containing
synthetic glycerol showed a slight dose related trend of increased
water consumption. Organ/body weight ratios of male and female rats
showed a significant increase in kidney ratio of rats maintained on
diets containing 20% glycerol, and of the heart of rats maintained on
diets containing 10% glycerol. Histopathological examination of organs
and tissues did not reveal compound or dose related effects (Atlas
Chemical Ind., 1969).

Groups each of 22 (Long-Evans strain) rats, equally divided by
sex were maintained on diets containing 5, 10 or 20% natural glycine
or synthetic glycerol (prepared from propylene). The control group
consisted of 26 rats (equally divided by sex). The 20% groups were
maintained for one year on the test diets, all other groups for two
years. Urinalysis of hematologic studies were made at 3 monthly
intervals during the first year of the study, then at 6 month
intervals. Urinalysis and hematologic parameters were similar for test
and control animals. Liver glycogen and fat content of the livers of
rats in the 20% group were similar for the natural and synthetic
glycerin groups. Organ/body ratios showed females receiving 20%
synthetic glycerin had liver/body ratios significantly higher than
controls and females receiving 5% synthetic glycerin had heart/body
ratios significantly greater than those on the 5% natural glycerin.
Microscopic examination of the liver, spleen, adrenals, kidney, small
intestine, bladder and reproductive organs showed no compound or dose
related effects (Hine et al., 1953).

Observations in Man

Fourteen (10 male, 4 female) graduate students ingested 110 g of
95% glycerol in 3 divided doses with their food daily for a period of
50 days. Preceding and following this period were 10 day control
periods. Uric acid excretion and basal metabolism were not
significantly affected, nor were there changes in red and white blood
cell counts or hemoglobin level during the test period. No adverse
effects were reported (Johnson, 1933).

Comments

Glycerol occurs naturally in fats and other substances which are
in part made up of lipid complexes. Glycerol may be derived from
natural sources, primarily triglycerides, or be synthesized by the
hydrogenolysis of carbohydrate materials or from products such as
propylene. Evidence is available to show that glycerol is metabolized
in the body to form glycogen or provide a direct energy source. In
addition, long-term studies are available to show that synthetically
derived glycerols are biologically similar to naturally derived
glycerol.

Because certain butanetriols can be contaminants of glycerol
produced by hydrogenolysis of carbohydrates, there is need to specify
maximum levels of such contaminants.

EVALUATION

ADI for man not specified.

    See Also:
       Toxicological Abbreviations