GLYCEROL ESTER OF WOOD ROSIN
(food and beverage grade)
First draft prepared by
Dr G.J.A. Speijers
Section on Public Health of the Advisory Centre of Toxicology,
National Institute of Public Health and Environmental Protection
(RIVM), Bilthoven, Netherlands
Explanation
Biological data
Comments
Evaluation
References
1. EXPLANATION
Glycerol esters of resin acids of wood rosins used as food
additives in beverages and chewing gum are those prepared from wood
rosin that is harvested from the stumps of the longleaf pine (Pinus
palustris) and purified to a beverage-grade ester gum. The resin
acid composition of wood rosin can vary considerably; however, the
main resin acids in ester gum are abietic acids, with smaller contents
of dehydroabietic and neoabietic acids; pimaric acids, including
isopimaric and sandaracopimaric acids; and palustric acid. The
toxicology of glycerol esters of wood rosins harvested from the stumps
of the pine tree is different from that of glycerol esters from
tall-oil and gums, which are not used for the preparation of food
additives. The latter are therefore not reviewed or evaluated in this
monograph addendum.
Resin acids of wood rosins can react on their carboxylic acid
group with salts, by esterification to ethylene glycol, diethylene
glycol, glycerol, and pentaerythrol, by reduction to alcohols and
aldehydes, and by decarboxylation; and also on their double bonds by
isomerization (to abietic, norabietic, palustric, and levopimaric
acids), by hydrogenation (dihydro- and tetrahydro- products), by
dehydrogenation or disproportionation (dehydroabietic and di- and
tetrahydro- products), Diels-Alder adducts (maleic anhydride and
fumaric acid), oxidation and polymerization. The carboxylic group of
the resin acids in wood rosin is attached to a tertiary carbon which
is sterically hindered. In order to esterify this type of hindered
carboxyl, higher temperatures and generally more drastic conditions
are used than for other carboxylic acids. These steric effects are
also responsible for the resistance of the resin acid ester linkage to
cleavage by water, acid, and alkali.
Glycerol ester of wood rosin was previously considered by the
Committee at its eighteenth, twentieth, thirty-third, and forty-fourth
meetings (Annex, references 35, 41, 83, and 116). At its twentieth
meeting, the Committee noted that in view of the stable ester bond and
the anticipated stability of this material, studies of long-term and
reproductive toxicity should be performed on the specific substance,
as opposed to unmodified resin, before further evaluation.
Specifications for food-grade material were adopted at the thirty-
seventh meeting of the Committee (Annex 1, references 94 and 96),
which described the material as a complex mixture of tri- and
diglycerol esters of resin acids from wood rosin with a residual
fraction of monoglycerol ester varying from 1 to 3%. At the forty-
fourth meeting of the Committee, a full monograph was prepared on
glycerol ester of wood rosin (Annex 1, reference 117). On the basis of
the results of studies of faecal excretion by rats of unlabelled
glycerol ester of wood rosin (identified as 'beverage-grade ester
gum'), the authors concluded that hydrolysis was minor; however, the
low sensitivity of the analytical chemical method used did not allow a
conclusion about the stability or non-bioavailability of glycerol
ester of wood rosin. The Committee was unable to establish an ADI
until studies became available demonstrating the metabolic stability
and non-bioavailability of glycerol ester of wood rosin under
conditions resembling those present in the human gastrointestinal
tract.
2. BIOLOGICAL DATA: Absorption, distribution and excretion
Ester gum 8BG was fed in the diet to groups of six male and six
female Fischer 344 rats either for 24 h at concentrations of 7000
or 28 000 mg/kg diet or for 10 days at concentrations of 14 000 or
28 000 mg/kg diet. Food consumption was measured during the treatment
period, and ester gum 8BG intake was calculated for each treatment
group. Faeces were collected during each 24-h treatment period and for
subsequent 24-h periods until no ester gum 8BG was detected.
Analytical validation showed that the limit of detection of ester gum
8BG in rat faeces was about 25 mg/ 7 g of faecal material. Studies of
the recovery of ester gum added to rat faecal samples resulted in
recoveries of 94-118 % of the nominal concentration, with an overall
relative standard deviation of less than 10%.
After dietary intake for 24 h, most of the ingested ester gum
was excreted in the faeces after 48-72 h. Of the dose of 7000 mg/kg,
75% was accounted for in the faeces, and total recovery at the
28 000 mg/kg dietary level was 95% of the amount ingested. The author
postulated that the lower recovery from the 7000 mg/kg diet was due to
the fact that the concentrations in faeces were near the detection
limit; the low recovery can thus be attributed to the lack of
sensitivity of the analytical method.
After repeated intake of ester gum 8BG over 10 days, total
recovery was about 102% of the amount ingested at the 14 000 mg/kg
dietary level and about 91% at the 28 000 mg/kg level. The author
concluded that at these doses, the total faecal recoveries were
essentially equal to the amounts ingested. As the mouth-to-anus
transit time in this experiment was not long, it was concluded that
enterohepatic cycling of ester gum 8BG did not occur and that no
measurable hydrolysis of ester gum 8BG took place in the rat intestine
(Blair, 1995).
In a pharmacokinetic study with [1,3-14C]glycerol ester gum
8BG, Fischer 344 rats received a single dose of about 200 mg/kg bw by
gavage after one (five male and five female rats) or 10 days (five
male rats) of dietary administration of unlabelled compound. The
degree of absorption of ester gum was determined by quantifying the
amount of radioactivity eliminated in expired carbon dioxide, urine,
and faeces during the 120-h interval after administration and by
assessing the residual radioactivity in the carcass 120 h after
treatment. In a separate investigation with five male rats, radiolabel
was determined in bile and blood at 4- and 12-h intervals for 24 h
after administration of 14C-ester gum. The extent of hydrolysis of
the ester gum was assessed by reverse-phase high-performance liquid
chromatography (HPLC) of extracts of faeces.
In both male and female rats fed ester gum for one day, 1% or
less of the administered radiolabel was excreted either as expired
carbon dioxide or in urine within 120 h. Most of the dose (> 95%) was
recovered in faeces and cage rinses. Only traces (< 0.2% of the
total dose) of radiolabel were detected in eight of 15 carcasses
obtained 120 h after treatment. According to the author, the traces
may have been unabsorbed 14C-ester gum remaining in the gut, since
the gastrointestinal tract was not removed before radioanalyses of the
carcasses. Similar results (< 1.1% in expired carbon dioxide or
urine) were obtained for male rats given the labelled ester gum after
a 10-day dietary administration of unlabelled ester gum. HPLC analysis
of the faeces of male rats collected during the first 48 h after
administration of 14C-ester gum showed that they contained a higher
percentage of a radioactive peak that eluted at the approximate void
volume of the column than did a standard solution of 14C-ester gum,
with 0.8% of the administered dose in samples collected at 0-12 h,
2.2% at 12-24 h, and 0.8% at 24-48 h after treatment. Two small peaks
of radioactivity that were present in 14C-ester gum and eluted at
the approximate retention time of monoglycerol esters of ester gum
were not detectable in faeces, indicating that only a very small
percentage of the administered 14C-ester gum, probably monoglycerol
esters, was hydrolysed.
In a separate study, five male rats with jugular vein and biliary
duct cannulas excreted 1.6-2.9 % of the dose into bile during a 24-h
interval after oral administration of 14C-ester gum. In HPLC
analyses conducted on two bile samples obtained 0-4 h after treatment,
all of the radioactivity eluted at the approximate void volume of the
column, indicating the presence of hydrolysed components, which may be
the same as those eliminated in faeces. The maximal total blood
content of radioactivity at 4, 8, 12, or 24 h after administration of
the labelled ester gum accounted for < 0.1% of the dose. In livers
collected from the same rats 24 h after treatment, radiolabel
represented 0.1-0.2% of the dose. These results indicate that only low
levels of radioactivity were absorbed; they also indicate that
14C-ester gum undergoes little hydrolysis or degradation in the
gastrointestinal tract. The author concluded that the metabolism of
14C-ester gum may involve hydrolysis of the monoglycerol esters
present in the formulation (Noker, 1996).
The possible metabolic fate of ester gum 8BG was also studied
in vitro. [1,3-14C]Glycerol ester gum 8BG was incubated at a
concentration of 4.4 or 0.5 mg/ml with human faecal extract, simulated
gastric fluid, or sterile water (as a negative control) for 24 h.
Samples collected 0, 6, and 24 h after incubation were analysed
in a HPLC-radiodetector system. The elution patterns on the
radiochromatogram were similar. Since ester gum is a mixture, further
analysis was based on changes in radioactivity in seven regions
related to the peaks of radioactivity on the radiogram. With the
negative control, no significant changes were observed at either
concentration. With human faecal extracts, occasional, minor
differences were seen in regions containing small peaks. With the low
concentration of ester gum in simulated gastric fluid, there was no
significant change in the peak regions after 24 h; however, with the
high concentration, a slight decrease was seen at 24 h in one peak
region which is associated with the triglycerides of rosin in
comparison with the 6-h sample; but there was no significant
difference in that peak region at 6 h relative to 0 h. Since the
emptying time of the stomach is normally about 4h, changes occurring
after that time will have no effect on the stability of ester gum in
the stomach in vivo. These studies showed no substantial change with
the longest incubation, indicating that beverage-grade ester gum is
stable in the human gastrointestinal tract (Tsu-Han Lin, 1996).
3. COMMENTS
The present Committee reviewed new studies with 14C-labelled
glycerol ester of wood rosin administered to rats and in vitro,
which indicate that the food-grade material is quite stable in the
gastrointestinal tract and that only a minor fraction, most likely the
monoglycerol ester fraction, undergoes partial hydrolysis. The
Committee therefore based the present evaluation on the new studies
showing the metabolic stability of glycerol ester of wood rosin and
the toxicological data available for food-grade and non-food-grade
material and wood rosin evaluated at the forty-fourth meeting (Annex
1, reference 116). From these studies, it was concluded that glycerol
ester of wood rosin has no genotoxic properties; wood rosin at doses
up to 434 mg/kg of body weight (bw) per day did not induce any
treatment-related histopathological changes in a long-term study of
toxicity and carcinogenicity in Sprague-Dawley rats; and the food-
grade material was less toxic than the non-food-grade material in
13-week studies of toxicity.
4. EVALUATION
Although there were no long-term studies of toxicity or
reproductive toxicity available, the Committee considered that the
data from previously reviewed studies and the new studies confirming
non-bioavailability were adequate to establish an ADI. Therefore, on
the basis of the 13-week toxicity study in rats with food-grade
material, in which the effect level was 2500 mg/kg bw per day, the
Committee allocated an ADI of 0-25 mg/kg bw, applying a safety factor
of 100. The Committee did not round the ADI to one significant figure,
because such rounding would have resulted in a decrease in the value
of the ADI of 20%.
5. REFERENCES
Blair, M. (1995) A dietary excretion study with Ester Gum 8BG in
Fischer 344 rats. Report No. 3352.2 from Springborn Laboratories,
Inc., Spencervile, OH, USA. Submitted to WHO by Hercules Inc.,
Wilmington,DE, USA.
Noker, P.E. (1996) Pharmacokinetic study of Ester Gum 8BG in rats.
Report project No. 8801 from Southern Research Institute, Birmingham,
AL 35205, USA. Submitted to WHO by ILSI North America, Washington DC,
USA.
Tsu-Han Lin (1996) Metabolism study of Ester Gum 8BG in human faecal
extracts and simulated human gastric juice. Report Project No. 8871
from Southern Research Institute, Birmingham, AL 35205, USA. Submitted
to WHO by ILSI North America, Washington DC, USA.