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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to GLP and contains the essential elements specified in OECD Guideline 417: Toxicokinetics.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to GLP and contains the essential elements specified in OECD Guideline 417: Toxicokinetics.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Reason / purpose:
read-across source
Details on absorption:
Absorption/Disposition of radioactivity (Phase I):
The results of the present study indicate that only a small percentage of the administered dose was absorbed by male rats given a single oral dose of approximately 200 mg/kg bw of [C^14]ester gum. Only a small percentage of the administered dose appeared to be hydrolyzed in the gastrointestinal tract.

The levels of radioactivity recovered in either expired CO2, urine or the cage rinses each accounted for only 1% or less of the dose. Since most of the administered radioactivity was recovered in feces, the radioactivity present in the cage rinses was assumed to be derived from fecal material. In carcasses collected at 120 hours after dose administration, only traces of radioactivity (0.2%) were detectable in 4 of the 5 carcasses analyzed. Since radioactivity was detectable in fecal samples collected from 96-120 hours post dose, it is believed that this trace amount was due to the gastrointestinal tract not being removed prior to analysis of the carcasses. It was assumed that the low levels of radioactivity present in the carcasses did not represent absorbed radioactivity but rather residual [C^14]ester gum in the intestines. Using the radioactivity excreted in expired CO2 and urine as an indication of [C^14]ester gum absorption, the results indicated that only 1.1-1.7% of the administered dose was absorbed by male rats. These results are consistent with the reported low toxicity of orally administered ester gum (Blair, 1991) and are similar to data which demonstrated that rats excreted dietary ester gum almost quantitatively in the feces (Blair, 1994).

References:
Blair M, 1991. 13-Week Dietary Toxicity Study in Rats. Unpublished report. International Research and Development Corporation (IRDC), Mattawan, MI. IRDC Study No. 548-007.

Blair N, 1994. A dietary excretion study with Ester Gum 8BG in Fischer 344 rats. Unpublished report. Springborn Laboratories, Inc. (SLS), Spencerville, OH. SLS Study No. 3352.1.

Absorption/Disposition of radioactivity (Phase II):
The disposition of radioactivity was similar to that observed for Phase I male rats which indicates that a 10-day dietary administration of ester gum did not affect the absorption and/or disposition of the test substance, as compared with 1-day dietary administration.

Absorption/Disposition of radioactivity (Phase III):
These results also indicate that only low levels of radioactivity were absorbed by rats following oral administration of a single dose of approximately 200 mg/kg bw of [C^14]ester gum and the radioactive species excreted in bile appeared to be a hydrolyzed product of [C^14]ester gum. Radioactivity was excreted in bile within 4 hours after dosing and was detectable in all samples collected through 24 hours post dose. The total amount of radioactivity excreted in bile during the 24-hour collection period ranged from 1.6-2.9% of the dose among the individual animals. HPLC analyses conducted on two bile samples collected from 0-4 hours after administration of [C^14]ester gum indicated that no intact [C^14]ester gum was present in the samples. Most of the radioactivity eluted as a single peak at the void volume suggesting that the radioactivity represented a hydrolysis product(s).

Low levels of radioactivity detected in blood during the first 24 hours accounted for 0.1% or less of the administered dose assuming that the total blood volume of each rat represented 9% of the body weight. In livers collected from these same rats at 24 hours after dosing, the amount of radioactivity per liver accounted for 0.1-0.2% of the administered dose indicating that hepatic retention of radioactivity was essentially negligible.

Absorption/Disposition of radioactivity (Phase IV):
The results indicated that, similar to male rats in Phase I, female rats absorbed less than 2% of the administered radioactivity within a 120-hour interval following oral administration of a single dose of approximately 200 mg/kg bw of [C^14]ester gum. Only 1% or less of the dose was recovered in either expired CO2, urine or the cage rinses. Like the male rats, the radioactivity present in the cage rinses was assumed to be derived from fecal material and not derived from absorbed radioactivity. Small traces of radioactivity were detectable in carcasses obtained at 120 hours after dosing and like Phase I males it is believed that this represented residual radioactivity in the gastrointestinal tract and not absorbed [C^14]ester gum.
Details on excretion:
Disposition of radioactivity (Phase I):
Between 94.7-105.7% of the dose was eliminated in the feces during the 120-hour sample collection interval. Of the total dose accounted for in the various sample types collected, 96.4-107.7% was eliminated within 48 hours after dose administration.

Disposition of radioactivity (Phase II):
The disposition of radioactivity was similar to that observed for Phase I male rats which indicates a majority of ester gum is eliminated in the feces within the first 48 hours after administration.

Disposition of radioactivity (Phase III):
The results of this experiment were consistent with the results obtained from Phases I, II, and IV in that a majority of ester gum ingestion is eliminated in the feces.

Disposition of radioactivity (Phase IV):
Of the total dose recovered (99.1-105.9%) from each female rat, 97.6-98.5% of the total was recovered in feces. As observed for Phase I male rats, female rats eliminated the majority of the dose within the first 48 hours after dosing.
Metabolites identified:
yes
Details on metabolites:
No metabolites were specifically identified in this study. However, radioanalyses of fecal extracts following oral administration of [C^14]ester gum indicate that little hydrolysis occurred and that 10-day dietary administration of ester gum did not affect the hydrolysis as compared with one-day dietary administration.

Metabolite identification (Phase I):
When results of HPLC analyses of the pooled THF extracts of feces collected from male rats were compared to a chromatogram obtained during the analysis of [C^14]ester gum, the elution pattern of radioactivity in the fecal extracts was essentially similar to that of the [C^14]ester gum formulation. The majority of the radioactivity in either sample type eluted at the approximate retention times of the di- and tri-esters. Only minor differences were found between the chromatograms of the fecal samples and the [C^14]ester gum. The feces samples contained a higher percentage of a radioactive peak which eluted at the approximate void volume of the column than did the [C^14]ester gum standard. The levels of this peak accounted for about 0.7% of the total radioactivity in the bulk formulation of [C^14]ester gum while the HPLC peak represented a mean value of 0.8%, 2.2%, and 0.8% of the total dose excreted in feces by the male rats at 0-12, 12-24, and 24-48 hours, respectively, after dosing. In addition, two small radioactive peaks were present in [C^14]ester gum and which eluted at the approximate retention time of mono-esters were not detectable in feces. This may have indicated that these radiolabelled components were hydrolyzed to the radioactive component(s) that eluted at the void volume. The results of the HPLC analysis of feces indicated that only a very small percentage of the administered [C^14]ester gum was hydrolyzed by rats. Further, the hydrolyzed species may have been the mono-ester of the compound.

Metabolite identification (Phase II):
The elution pattern of radioactivity was similar to that of Phase I males where the mean percentages accounted for approximately 2.2% of the total administered dose in feces collected 12-24 hours and 1.3% in feces collected 24-48 hours post dose. As observed after 1-day dietary administration of ester gum, little hydrolysis of [C^14]ester gum occurred in male rats given ester gum in the diet for 10 days prior to administration of the radiolabelled compound. Similarly, fecal extracts from the 10-day diet rats did not contain detectable amounts of radioactivity which eluted at the approximate retention time of the mono-esters indicating that 10-day dietary administration of ester gum did not affect the hydrolysis of [C^14]ester gum.

Metabolite identification (Phase III):
HPLC analyses conducted on two bile samples collected from 0-4 hours after administration of [C^14] ester gum indicated that no intact [C^14] ester gum was present in the samples; most of the radioactivity eluted as a single peak at the void volume of the column, suggesting that the radioactivity represented a hydrolysis product(s). This presumed hydrolysis product may have been the same product that was resolved in feces.

Test substance radioactivity and stability:

The results of the HPLC analysis of the bulk formulation of [C^14]ester gum prior to the initiation of the study indicated that the compound was 99.97% radiochemically pure and reanalysis of the radiolabelled compound at study termination yielded similar results. In turn the radiolabelled compound was considered stable. The chemical and radiochemical stability of ester gum in the dosing emulsions of [C^14]ester gum was determined by SEC analyses of an emulsion on the day of preparation and 3 days later and the results indicated that no chemical or radiochemical degradation of the ester gum in the emulsion occurred during the 3-day storage period.

 

Formulation analysis of [C^14]ester gum dosing emulsions:

Chemical concentration analyses and radioanalyses conducted on the top, middle and bottom samples indicated that each emulsion was homogeneous.

 

Formulation analysis of feed containing unlabelled ester gum:

The mean concentration of ester gum in the feed was comparable on Day 1 (16.7 mg/g) and Day 14 (16.4 mg/g) indicating that the ester gum had remained stable during this period.

 

Dietary consumption of ester gum (Phase I):

Each male rat consumed an average 207.6 mg of ester gum during the approximate 20-hour feeding period.

 

Dietary consumption of ester gum (Phase II):

Rats consumed a daily average of 124.6-252.9 mg of ester gum during the 10-day feeding period. 

 

Dietary consumption of ester gum (Phase IV):

Each female rat consumed an average of 139.7 mg of ester gum during the approximate 18-hour feeding period.

Conclusions:
Interpretation of results: low bioaccumulation potential based on study results
To investigate the absorption of orally administered ester gum, male and female rats were administered a single oral dose of approximately 200 mg/kg bw of [C^14]ester gum following one or ten days of dietary administration of the unlabelled compound. The degree of absorption of ester gum was determined by quantitating the amount of radioactivity eliminated in expired air, urine and feces during a 120-hour interval following oral administration of [C^14]ester gum and by assessing residual radioactivity in the carcasses at study termination. In a separate study, the amount of radioactivity excreted in bile and present in blood at 4 or 12-hour intervals through 24 hours after administration of [C^14]ester gum was determined. After dietary administration of Ester Gum 8BG at a dose concentration of 14,000 ppm for one to 10 days followed by a single oral gavage dose of 200 mg/kg/bw [C^14]ester gum, less than 5% ester gum was absorbed by each animal. Most of the dose (>95%) was recovered in feces and the cage rinses. Low levels of radioactivity recovered in some of the carcasses were attributed to unabsorbed [C^14]ester gum remaining in the gut. HPLC analyses of fecal and bile extracts indicated that only a very small percentage of the administered [C^14]ester gum was hydrolyzed. Radioactivity measured in the bile and the blood was less than 3% of the total radioactivity detected.

This study showed that following oral administration of [C^14]ester gum to rats, only low levels of radioactivity are absorbed. In addition, [C^14]ester gum undergoes only minor metabolism in the gastrointestinal tract. The metabolism of [C^14]ester gum may involve hydrolysis of the mono-esters present in the gum formulation.
Executive summary:

This data is being read across from the source study that tested Resin acids and Rosin acids, esters with glycerol based on category read across that is explained in the category justification document attached in Section 13 of the dossier.

In a study to investigate the absorption of orally administered ester gum, groups of 8 males and 8 females were provided ad libitum access to diets containing14000 ppm unlabelled Ester Gum 8BG for approximately 1 day, 8 males were provided the same diet for 10 days, and a separate group of 9 males received no dietary Ester Gum 8BG but were implanted with bile and jugular cannulas. After dietary administration of Ester Gum 8BG or after cannula insertion, each animal received a single oral gavage dose of 200 mg/kg/bw [C^14]ester gum. All animals were placed in glass metabolism cages. Those receiving dietary ester gum prior to administration of the labeled material had urine, feces, and expired CO2 collected every 12 hours during the first 24 hours and then every 24 hours for the entire 120-hour post-dose observation period. Animals with cannulas had bile and blood samples collected every 4 hours for 24 hours after administration of radiolabelled ester gum. 

 

In males and females exposed to unlabelled Ester Gum 8BG in the diet for one to 10 days prior to oral gavage administration of [C^14]ester gum, the levels of radioactivity recovered in either expired CO2, urine or the cage rinses each accounted for only 1% or less of the dose. Between 94.7-105.9% (males) and 97.6-98.5% (females) of the dose was eliminated in the feces during the 120-hour sample collection interval and the radioactivity present in the cage rinses was assumed to be derived from fecal material. Traces of radioactivity in the carcasses collected at study termination were believed to have been due to the gastrointestinal tract not being removed prior to analysis of the carcasses and it was assumed that the low levels of radioactivity present did not represent absorbed radioactivity but rather residual [C^14]ester gum in the intestines. HPLC analysis indicated that a small amount (2.2% or less) of the radiolabelled components were hydrolyzed by-products of [C^14]ester gum. Radioactivity analyses of two bile samples collected from 0-4 hours after administration of [C^14]ester gum indicated that no intact [C^14]ester gum was present in the samples. Most of the radioactivity (1.6-2.9%) eluted as a single peak suggesting that the radioactivity represented a hydrolysis product(s). Low levels of radioactivity detected in the blood during the first 24 hours after [C^14]ester gum administration accounted for 0.1% or less of the dose and radioactivity in livers from these same rats accounted for 0.1-0.2% of the administered dose indicating that hepatic retention of radioactivity was essentially negligible.

 

Based on the findings of the present study, ingestion of Ester Gum 8BG has a low potential to bioaccumulate. Only a small percentage of the administered [C^14]ester gum was hydrolyzed and absorbed from the gastrointestinal tract with most of the dose (>95%) recovered in the feces within the first 48 hours.  

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996

Materials and methods

Objective of study:
other: pharmacokinetics
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The method contained the essential elements outlined in OECD Guideline 417.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance (unlabelled):
-Test substance (as cited in report): Ester Gum 8BG glycerol ester of wood rosin
-Lot number: GGS-9057
-Supplier: Hercules Incorporated, Brunswick, GA, USA
-Storage: stored at approximately –20 °C under a nitrogen atmosphere

Test substance (radiolabelled):
-Preparation: The radiolabelled formulation was prepared using 1,3-[C^14] glycerol.
-Supplier: Sigma Chemical Company, St. Louis, MO, USA
-Specific activity: 2.5 mCi/g
-Storage: 5 °C
-Purity: 99.97%
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Test animals:
-Source: Charles River Laboratories, Raleigh, NC, USA
-Sex: Male and Female
-Number of animals: Male (25) and Female (8)
-Age at receipt: Male: 7-10 weeks; Female: 13-14 weeks
-Age at study initiation: Males: 9-11 weeks; Females: 15 weeks old
-Acclimation period: 10 days
-Housing (during quarantine): The animals were housed separately by sex, with a maximum of five animals per cage.
-Housing (during testing): During Phases I, II, and IV, male and female rats were group housed (4 rats/cage) by sex while receiving unlabelled test material. After administration of the radiolabelled test substance the animals were individually housed in glass metabolism cages where air was drawn through the cages continuously by a vacuum pump and then bubbled through two towers in series containing 5M ethanolamine in 2-methoxyethanol to fix expired CO2.
-Diet (during quarantine): NIH 07 meal (Zeigler Brothers, Gardners, PA), ad libitum
-Diet (during testing): Phase I, II, and IV animals received the formulated diet for 18 hr to 10 days. Once animals were placed in individual metabolism cages, plain NIH 07 meal was again provided ad libitum.
-Water (during quarantine): local municipality, ad libitum
-Water (during testing): Not stated but presumed to be the same as during the quarantine period while animals received unlabelled test material. Following radiolabelled test material administration, water was supplied ad libitum via water bottles attached to metabolism cages.
-Method of animal identification: individual numbered tail tattoos
-Method of animal distribution: Male rats were randomly assigned, using a table of random numbers, to individual phases of the study. Randomization of female rats was not performed since all females were assigned to the same dose group.
-Animal care: Animal care was conducted in accordance with the guidelines specified in "Guide for Care and Use of Laboratory Animals"

Environmental conditions: No data available.

Administration / exposure

Route of administration:
other: oral: feed and oral: gavage
Vehicle:
other: Oral: gavage- gum arabic mucilage preparation with an oil blend; Oral: feed- NIH 07 meal (Zeigler Brothers, Gardners, PA, USA)
Details on exposure:
The animals were divided into 4 groups:

Phase I:
Eight males were fed the formulated diet containing unlabelled ester gum for approximately 20 hours prior to the administration of [C^14]ester gum. On the day of dosing with [C^14]ester gum, five rats were appropriately dosed per os with the radiolabelled emulsion and excreta samples were collected from these animals; 2 rats were improperly dosed. The two improperly dosed male rats and the one remaining male rat were euthanized by CO2 inhalation.

Phase II:
Eight male rats were fed the formulated diet containing ester gum for 10 days prior to the administration of [C^14]ester gum. On the day of dosing with [C^14]ester gum, five rats were appropriately dosed per os with the radiolabelled emulsion and excreta samples were collected from these animals. The remaining 3 male rats were euthanized by CO2 inhalation.

Phase III:
Nine male rats were implanted with jugular vein cannulas and the following day, six of these rats were also implanted with biliary cannulas. The six rats implanted with two cannulas were then dosed by oral gavage with [C^14]ester gum and samples were collected from these animals at specified intervals. The rats which were not dosed were euthanized by CO2 inhalation.

Phase IV:
The eight female rats were fed the formulated diet containing ester gum for approximately 18 hours prior to the administration of [C^14]ester gum. On the day of dosing with [C^14]ester gum, five rats were dosed by oral gavage with the radiolabelled emulsion and excreta samples were collected from these animals. The remaining female rats were euthanized by CO2 inhalation.
Duration and frequency of treatment / exposure:
Dietary exposure: ad libitum for 18 hours -10 days followed by administration of a single dose of radiolabelled test material administered by oral gavage.

Sample collection following administration of radiolabelled test substance: 120 hours

Doses / concentrations
Remarks:
Doses / Concentrations:
Dietary exposure (Phases I, II, and IV): 14000 ppm

Oral gavage of radiolabelled material: 200 mg/kg bw
No. of animals per sex per dose:
Phase I: 8 males
Phase II: 8 males
Phase III: 9 males
Phase IV: 8 females
Control animals:
no
Details on study design:
Overview
The absorption of orally administered Ester Gum 8BG was investigated in male and female Fischer 344 rats orally gavaged with a single dose of approximately 200 mg/kg of [C^14]ester gum after one or ten days of dietary administration of the unlabelled compound (1400 ppm). The degree of absorption was determined by quantitating the amount of radioactivity eliminated in expired CO2, urine and feces during a 120-hour interval after dose administration and by assessing the residual radioactivity in the carcasses at 120 hours post dose. In a separate investigation, the amount of radioactivity excreted in bile and present in blood at 4 or 12-hour intervals through 24 hours after administration of [C^14]ester gum was determined. The extent of hydrolysis of orally administered [C^14]ester gum was assessed by chromatographic analysis, using reversed phase HPLC of extracts of collected feces and bile.

Formulated feed consumption:
During the period of formulated diet administration (Phases I and IV), rats were group housed (4 rats/cage) by sex and feed consumption was measured by obtaining the weight of each feeder at the beginning and end of the dietary administration period. The food consumption per rat was calculated by dividing the total feed consumed by the number of rats per cage and ester gum intake per rat was computed from the food consumption of each rat and the mean concentration of ester gum in the diet. In Phase II, rats were again group-housed (4 rats/cage) but fresh formulated feed was supplied each day. Daily food consumption was measured and calculated as above.

Radiolabelled dosing and sample collection (Phase I, II, and IV):
Immediately after dosing with 200 mg/kg bw of [14^C]ester gum, each rat was placed in individual glass metabolism cages where air was drawn through the cages continuously by a vacuum pump and then bubbled through two towers in series containing 5M ethanolamine in 2-methoxyethanol to fix expired CO2. Feed (NIH 07 Blox, Zeigler Brothers) and water were supplied ad libitum. Expired CO2, urine and feces samples were collected from each animal at 0-12 hour, 12-24 hour and subsequent 24-hour intervals through 120 hours after dosing. At each collection interval, the inside of the metabolism cages was rinsed with water and each rinse was saved for analysis. At the end of the sample collection period, rats were euthanized by CO2 asphyxiation and the carcasses saved.

Sample analysis:
The volume of each urine, cage rinse and CO2 tower solution was measured. Duplicate portions (0.3-1.0 mL) of individual urine samples were placed in scintillation vials and decolorized with 0.5 mL of 30% hydrogen peroxide. One milliliter duplicate portions of each cage rinse and tower solution were placed in scintillation vials. Feces were weighed and homogenized in 9 volumes of THF and quadruplicate portions (0.5 mL) of each THF supernate were placed in individual scintillation vials. Carcasses were weighed and homogenized in 9 volumes of water; quadruplicate portions of approximately 1.0 mL were placed in scintillation vials and solubilized with Soluene tissue solubilizer. Each scintillation vial was subsequently radioassayed after the addition of scintillator.

Feces samples collected from male rats (Phases I and II) during the first 48 hours after administration of [C^14]ester gum were also analyzed by HPLC. Prior to analysis, equal volumes (1 mL) of individual fecal homogenates were pooled by time interval, centrifuged to precipitate solid material and then filtered through a 0.45 micron PTFE filter.

Sample storage (Phases I, II, and IV):
Urine, CO2, tower solution and cage rinse samples were maintained at approximately 5 ºC from the time of collection until the time of preparation for radioanalysis; thereafter, samples were stored at or below -20 ºC. Feces and carcasses were frozen upon collection; after preparation for radioanalysis, homogenates were maintained at or below -20 ºC.

Radiolabelled dosing and sample collection (Phase III):
Male rats were implanted with an indwelling jugular vein cannula while under sodium pentobarbital anesthesia and the following day biliary cannulas were placed in the animals following anesthesia with ketamine/xylazine/promazine, given i.p. After recovery from anesthesia, six rats were dosed per os with a single dose of approximately 200 mg/kg of [C^14]ester gum. Excreted bile was collected continuously and samples were obtained at 4, 8, 12, and 24 hours after dosing. Blood samples were obtained from the jugular cannula at the same time intervals with heparin being used as an anticoagulant. At 24 hours after dosing, each rat was exsanguinated while under CO2 anesthesia and the liver, kidney, spleen, mesenteric lymph nodes, perirenal fat and entire gastrointestinal tract (from mouth to anus) were removed from each animal. The residual carcasses were saved.

Sample analysis:
Duplicate portions of each bile sample (0.2 mL) and individual blood samples (0.1 mL) were placed in scintillation vials and decolorized with 30% hydrogen peroxide. The blood samples were subsequently digested with Soluene 350 tissue solubilizer. The samples were radioassayed after the addition of scintillator. Livers were weighed and homogenized in 9 volumes of deionized water and duplicate portions (1 mL) of each homogenate were placed in scintillation vials, digested with Soluene 350 tissue solubilizer and radioassayed after the addition of scintillator. Other collected tissues and the carcasses were saved but not analyzed.

Sample storage (Phase III):
Bile and blood samples were maintained at approximately 5 ºC prior to radioanalysis and for several weeks thereafter; samples were then transferred to storage at approximately -20 ºC. Tissues and carcasses were maintained at or below -15 ºC.
Details on dosing and sampling:
Radiolabelled purity:
The radiochemical purity of the bulk formulation of [C^14]ester gum was assessed by size exclusion chromatography (SEC).

Stability of radiolabelled formulation:
The stability of the bulk formulation of [C^14]ester gum was assessed by determining the radiochemical purity of the compound prior to and at the completion of the study using SEC analysis.

Stability of dosing emulsion:
The stability of [14^C]ester gum in the dosing emulsion was assessed by analyzing [14^C]ester gum emulsion for chemical concentration and radiochemical purity on the day of preparation and 3 days after preparation.

Pulverization of Ester Gum:
Pre-weighed quantities of unlabelled ester gum were placed in a mortar, liquid nitrogen was added and the frozen ester gum then was ground into a fine powder using a pestle. The powder subsequently was passed through a 60-mesh sieve and the pulverized ester gum was stored at –20 °C under a nitrogen atmosphere.

Preparation of Formulated Feed Containing Unlabelled Ester Gum:
Formulated feed containing unlabelled ester gum at a dose concentration of 14,000 ppm (14 mg/g) was prepared as follows: 4930 g of pre-chilled, NIH-07 meal (Zeigler Brothers, Gardners, PA) was weighed into a double thickness plastic bag. A premix was prepared where a portion (approximately 70 g) of the pre-weighed feed was mixed with 70 g of unlabelled ester gum until visually homogeneous. Increasing amounts of feed were added, with mixing after each addition, until the total amount of premix was 1000 g. The premix was then added to a V-shaped blender containing approximately half of the pre-weighed feed and the remaining pre-weighed feed was added to the top of the blender. The feed was blended for approximately 5 min with the intensifier bar on and approximately 10 min with the intensifier bar off. The formulated feed was stored at –20 °C under a nitrogen atmosphere.

Preparation of [C^14]ester gum emulsions:
Dosing formulations for oral gavage administration of ester gum were prepared as emulsions by mixing a gum arabic mucilage preparation with an oil blend containing both unlabelled and labelled [C^14]ester gum. To prepare gum arabic mucilage, gum arabic was hydrated with deionized water (1.35 g gum arabic and 6.4 g of water). Oil blends were prepared by mixing 0.15 g of [C^14]ester gum, 0.85 g of unlabelled ester gum and 3.38 g of com oil. The ester gum formulations and corn oil were stirred until complete solution of the ester gum was obtained. Dosing emulsions were prepared to contain 20 mg/g (mL) of ester gum with a radioactivity content of approximately 8 µCi/g (mL).

For preparation of each dosing emulsion, mucilage preparation and oil blend were mixed in the following proportions: 7.3 g of mucilage preparation and 0.7 g of oil blend. An emulsion was prepared by blending the mucilage and oil mixtures together until the particle size of the emulsion was 1 micron or less. Particle size was determined using a Coulter Model N4 MD Sub-Micron Particle Analyzer (Coulter Scientific Instruments, Hialeah, FL). With one exception, gum arabic mucilage preparations and oil blends were prepared on the day prior to preparation of each dosing emulsion. Oil blends were stored refrigerated; the mucilage preparations were maintained at room temperature. Dosing emulsions were prepared immediately prior to use and maintained at room temperature before administration. The volume of [C^14]ester gum dosing emulsion administered to each rat was 1 mL/kg bw.

Formulation Analysis:
-Formulated feed containing unlabelled ester gum:
Six samples of approximately 100 g each were removed from the bulk quantity of formulated feed containing ester gum. Homogeneity determinations were made on three samples (upper left, upper right, and bottom middle) removed from the V-shaped blender in which the feed was prepared. Two additional samples were removed for stability determinations and one sample was removed for archiving. Each formulated feed sample was placed in an individual plastic bag and feed samples analyzed on the day of feed preparation were maintained at room temperature prior to analysis. Stability samples were stored at –20 °C under nitrogen.

Prior to analysis, 5 or 10 g quantities of each formulated feed sample were weighed, placed in screw-capped bottles and mixed with 50 or 100 mL, respectively, of tetrahydrofuran (THF) containing 250 ppm BHT. The mixtures were maintained at room temperature overnight and portions of each THF extract were then filtered through 0.45 micron PTFE filters. Ester gum standards, containing approximately 3.5 mg/g to 28 mg/g of ester gum, were prepared by adding weighed quantities of unlabelled ester gum to unformulated feed and were extracted with 10 vol of THF. Each THF extract was assayed by SEC with a UV absorbance of 254 nm.

-[C^14]Ester gum emulsions:
Upon preparation, portions of the top, middle and bottom of each [C^14]ester gum dosing emulsion were removed for subsequent analysis. The following three analyses were conducted:
*Chemical concentration verification: Prior to analysis, duplicate portions of the top, middle and bottom samples were diluted 1:40 with THF. Ester gum standards, containing from 0.25 to 1 mg/mL of unlabelled ester gum, were prepared in THF containing 250 ppm of BHT. Each sample was analyzed by SEC with UV absorbance only.
*Radiolabelled purity determinations: These were conducted on the middle emulsion sample only. The sample was diluted into THF and then analyzed with SEC and the recovery of injected radioactivity was determined by collection and radioanalysis of the column effluent.
*Specific activity determinations: These were performed on the duplicate portions of 1:40 dilutions prepared in THF, of the top, middle and bottom samples of each dosing emulsion. The total radioactivity per mL of [C^14]ester gum dosing emulsion was determined after correction for dilution and volume radioassayed.
Statistics:
Mean values and standard deviations were computed using Lotus 1,2,3, version 2.3. No statistical analyses were performed.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Absorption/Disposition of radioactivity (Phase I):
The results of the present study indicate that only a small percentage of the administered dose was absorbed by male rats given a single oral dose of approximately 200 mg/kg bw of [C^14]ester gum. Only a small percentage of the administered dose appeared to be hydrolyzed in the gastrointestinal tract.

The levels of radioactivity recovered in either expired CO2, urine or the cage rinses each accounted for only 1% or less of the dose. Since most of the administered radioactivity was recovered in feces, the radioactivity present in the cage rinses was assumed to be derived from fecal material. In carcasses collected at 120 hours after dose administration, only traces of radioactivity (0.2%) were detectable in 4 of the 5 carcasses analyzed. Since radioactivity was detectable in fecal samples collected from 96-120 hours post dose, it is believed that this trace amount was due to the gastrointestinal tract not being removed prior to analysis of the carcasses. It was assumed that the low levels of radioactivity present in the carcasses did not represent absorbed radioactivity but rather residual [C^14]ester gum in the intestines. Using the radioactivity excreted in expired CO2 and urine as an indication of [C^14]ester gum absorption, the results indicated that only 1.1-1.7% of the administered dose was absorbed by male rats. These results are consistent with the reported low toxicity of orally administered ester gum (Blair, 1991) and are similar to data which demonstrated that rats excreted dietary ester gum almost quantitatively in the feces (Blair, 1994).

References:
Blair M, 1991. 13-Week Dietary Toxicity Study in Rats. Unpublished report. International Research and Development Corporation (IRDC), Mattawan, MI. IRDC Study No. 548-007.

Blair N, 1994. A dietary excretion study with Ester Gum 8BG in Fischer 344 rats. Unpublished report. Springborn Laboratories, Inc. (SLS), Spencerville, OH. SLS Study No. 3352.1.

Absorption/Disposition of radioactivity (Phase II):
The disposition of radioactivity was similar to that observed for Phase I male rats which indicates that a 10-day dietary administration of ester gum did not affect the absorption and/or disposition of the test substance, as compared with 1-day dietary administration.

Absorption/Disposition of radioactivity (Phase III):
These results also indicate that only low levels of radioactivity were absorbed by rats following oral administration of a single dose of approximately 200 mg/kg bw of [C^14]ester gum and the radioactive species excreted in bile appeared to be a hydrolyzed product of [C^14]ester gum. Radioactivity was excreted in bile within 4 hours after dosing and was detectable in all samples collected through 24 hours post dose. The total amount of radioactivity excreted in bile during the 24-hour collection period ranged from 1.6-2.9% of the dose among the individual animals. HPLC analyses conducted on two bile samples collected from 0-4 hours after administration of [C^14]ester gum indicated that no intact [C^14]ester gum was present in the samples. Most of the radioactivity eluted as a single peak at the void volume suggesting that the radioactivity represented a hydrolysis product(s).

Low levels of radioactivity detected in blood during the first 24 hours accounted for 0.1% or less of the administered dose assuming that the total blood volume of each rat represented 9% of the body weight. In livers collected from these same rats at 24 hours after dosing, the amount of radioactivity per liver accounted for 0.1-0.2% of the administered dose indicating that hepatic retention of radioactivity was essentially negligible.

Absorption/Disposition of radioactivity (Phase IV):
The results indicated that, similar to male rats in Phase I, female rats absorbed less than 2% of the administered radioactivity within a 120-hour interval following oral administration of a single dose of approximately 200 mg/kg bw of [C^14]ester gum. Only 1% or less of the dose was recovered in either expired CO2, urine or the cage rinses. Like the male rats, the radioactivity present in the cage rinses was assumed to be derived from fecal material and not derived from absorbed radioactivity. Small traces of radioactivity were detectable in carcasses obtained at 120 hours after dosing and like Phase I males it is believed that this represented residual radioactivity in the gastrointestinal tract and not absorbed [C^14]ester gum.
Details on excretion:
Disposition of radioactivity (Phase I):
Between 94.7-105.7% of the dose was eliminated in the feces during the 120-hour sample collection interval. Of the total dose accounted for in the various sample types collected, 96.4-107.7% was eliminated within 48 hours after dose administration.

Disposition of radioactivity (Phase II):
The disposition of radioactivity was similar to that observed for Phase I male rats which indicates a majority of ester gum is eliminated in the feces within the first 48 hours after administration.

Disposition of radioactivity (Phase III):
The results of this experiment were consistent with the results obtained from Phases I, II, and IV in that a majority of ester gum ingestion is eliminated in the feces.

Disposition of radioactivity (Phase IV):
Of the total dose recovered (99.1-105.9%) from each female rat, 97.6-98.5% of the total was recovered in feces. As observed for Phase I male rats, female rats eliminated the majority of the dose within the first 48 hours after dosing.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
No metabolites were specifically identified in this study. However, radioanalyses of fecal extracts following oral administration of [C^14]ester gum indicate that little hydrolysis occurred and that 10-day dietary administration of ester gum did not affect the hydrolysis as compared with one-day dietary administration.

Metabolite identification (Phase I):
When results of HPLC analyses of the pooled THF extracts of feces collected from male rats were compared to a chromatogram obtained during the analysis of [C^14]ester gum, the elution pattern of radioactivity in the fecal extracts was essentially similar to that of the [C^14]ester gum formulation. The majority of the radioactivity in either sample type eluted at the approximate retention times of the di- and tri-esters. Only minor differences were found between the chromatograms of the fecal samples and the [C^14]ester gum. The feces samples contained a higher percentage of a radioactive peak which eluted at the approximate void volume of the column than did the [C^14]ester gum standard. The levels of this peak accounted for about 0.7% of the total radioactivity in the bulk formulation of [C^14]ester gum while the HPLC peak represented a mean value of 0.8%, 2.2%, and 0.8% of the total dose excreted in feces by the male rats at 0-12, 12-24, and 24-48 hours, respectively, after dosing. In addition, two small radioactive peaks were present in [C^14]ester gum and which eluted at the approximate retention time of mono-esters were not detectable in feces. This may have indicated that these radiolabelled components were hydrolyzed to the radioactive component(s) that eluted at the void volume. The results of the HPLC analysis of feces indicated that only a very small percentage of the administered [C^14]ester gum was hydrolyzed by rats. Further, the hydrolyzed species may have been the mono-ester of the compound.

Metabolite identification (Phase II):
The elution pattern of radioactivity was similar to that of Phase I males where the mean percentages accounted for approximately 2.2% of the total administered dose in feces collected 12-24 hours and 1.3% in feces collected 24-48 hours post dose. As observed after 1-day dietary administration of ester gum, little hydrolysis of [C^14]ester gum occurred in male rats given ester gum in the diet for 10 days prior to administration of the radiolabelled compound. Similarly, fecal extracts from the 10-day diet rats did not contain detectable amounts of radioactivity which eluted at the approximate retention time of the mono-esters indicating that 10-day dietary administration of ester gum did not affect the hydrolysis of [C^14]ester gum.

Metabolite identification (Phase III):
HPLC analyses conducted on two bile samples collected from 0-4 hours after administration of [C^14] ester gum indicated that no intact [C^14] ester gum was present in the samples; most of the radioactivity eluted as a single peak at the void volume of the column, suggesting that the radioactivity represented a hydrolysis product(s). This presumed hydrolysis product may have been the same product that was resolved in feces.

Any other information on results incl. tables

Test substance radioactivity and stability:

The results of the HPLC analysis of the bulk formulation of [C^14]ester gum prior to the initiation of the study indicated that the compound was 99.97% radiochemically pure and reanalysis of the radiolabelled compound at study termination yielded similar results. In turn the radiolabelled compound was considered stable. The chemical and radiochemical stability of ester gum in the dosing emulsions of [C^14]ester gum was determined by SEC analyses of an emulsion on the day of preparation and 3 days later and the results indicated that no chemical or radiochemical degradation of the ester gum in the emulsion occurred during the 3-day storage period.

 

Formulation analysis of [C^14]ester gum dosing emulsions:

Chemical concentration analyses and radioanalyses conducted on the top, middle and bottom samples indicated that each emulsion was homogeneous.

 

Formulation analysis of feed containing unlabelled ester gum:

The mean concentration of ester gum in the feed was comparable on Day 1 (16.7 mg/g) and Day 14 (16.4 mg/g) indicating that the ester gum had remained stable during this period.

 

Dietary consumption of ester gum (Phase I):

Each male rat consumed an average 207.6 mg of ester gum during the approximate 20-hour feeding period.

 

Dietary consumption of ester gum (Phase II):

Rats consumed a daily average of 124.6-252.9 mg of ester gum during the 10-day feeding period. 

 

Dietary consumption of ester gum (Phase IV):

Each female rat consumed an average of 139.7 mg of ester gum during the approximate 18-hour feeding period.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: low bioaccumulation potential based on study results
To investigate the absorption of orally administered ester gum, male and female rats were administered a single oral dose of approximately 200 mg/kg bw of [C^14]ester gum following one or ten days of dietary administration of the unlabelled compound. The degree of absorption of ester gum was determined by quantitating the amount of radioactivity eliminated in expired air, urine and feces during a 120-hour interval following oral administration of [C^14]ester gum and by assessing residual radioactivity in the carcasses at study termination. In a separate study, the amount of radioactivity excreted in bile and present in blood at 4 or 12-hour intervals through 24 hours after administration of [C^14]ester gum was determined. After dietary administration of Ester Gum 8BG at a dose concentration of 14,000 ppm for one to 10 days followed by a single oral gavage dose of 200 mg/kg/bw [C^14]ester gum, less than 5% ester gum was absorbed by each animal. Most of the dose (>95%) was recovered in feces and the cage rinses. Low levels of radioactivity recovered in some of the carcasses were attributed to unabsorbed [C^14]ester gum remaining in the gut. HPLC analyses of fecal and bile extracts indicated that only a very small percentage of the administered [C^14]ester gum was hydrolyzed. Radioactivity measured in the bile and the blood was less than 3% of the total radioactivity detected.

This study showed that following oral administration of [C^14]ester gum to rats, only low levels of radioactivity are absorbed. In addition, [C^14]ester gum undergoes only minor metabolism in the gastrointestinal tract. The metabolism of [C^14]ester gum may involve hydrolysis of the mono-esters present in the gum formulation.
Executive summary:

In a study to investigate the absorption of orally administered ester gum, groups of 8 males and 8 females were provided ad libitum access to diets containing14000 ppm unlabelled Ester Gum 8BG for approximately 1 day, 8 males were provided the same diet for 10 days, and a separate group of 9 males received no dietary Ester Gum 8BG but were implanted with bile and jugular cannulas. After dietary administration of Ester Gum 8BG or after cannula insertion, each animal received a single oral gavage dose of 200 mg/kg/bw [C^14]ester gum. All animals were placed in glass metabolism cages. Those receiving dietary ester gum prior to administration of the labeled material had urine, feces, and expired CO2 collected every 12 hours during the first 24 hours and then every 24 hours for the entire 120-hour post-dose observation period. Animals with cannulas had bile and blood samples collected every 4 hours for 24 hours after administration of radiolabelled ester gum. 

 

In males and females exposed to unlabelled Ester Gum 8BG in the diet for one to 10 days prior to oral gavage administration of [C^14]ester gum, the levels of radioactivity recovered in either expired CO2, urine or the cage rinses each accounted for only 1% or less of the dose. Between 94.7-105.9% (males) and 97.6-98.5% (females) of the dose was eliminated in the feces during the 120-hour sample collection interval and the radioactivity present in the cage rinses was assumed to be derived from fecal material. Traces of radioactivity in the carcasses collected at study termination were believed to have been due to the gastrointestinal tract not being removed prior to analysis of the carcasses and it was assumed that the low levels of radioactivity present did not represent absorbed radioactivity but rather residual [C^14]ester gum in the intestines. HPLC analysis indicated that a small amount (2.2% or less) of the radiolabelled components were hydrolyzed by-products of [C^14]ester gum. Radioactivity analyses of two bile samples collected from 0-4 hours after administration of [C^14]ester gum indicated that no intact [C^14]ester gum was present in the samples. Most of the radioactivity (1.6-2.9%) eluted as a single peak suggesting that the radioactivity represented a hydrolysis product(s). Low levels of radioactivity detected in the blood during the first 24 hours after [C^14]ester gum administration accounted for 0.1% or less of the dose and radioactivity in livers from these same rats accounted for 0.1-0.2% of the administered dose indicating that hepatic retention of radioactivity was essentially negligible.

 

Based on the findings of the present study, ingestion of Ester Gum 8BG has a low potential to bioaccumulate. Only a small percentage of the administered [C^14]ester gum was hydrolyzed and absorbed from the gastrointestinal tract with most of the dose (>95%) recovered in the feces within the first 48 hours.