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
Link to IPCS INCHEM

• EXPLANATION
• BIOLOGICAL DATA
• COMMENTS
• VALUATION
• 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 istopathological 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, pplying 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.