Sucrose, mixed acetate and isobutyrate octaesters

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

α-​D-​Glucopyranoside, β-​D-​fructofuranosyl, diacetate hexakis(2-​methylpropanoate)

Cas Number:

27216-37-1

Molecular formula:

C40H62O19

IUPAC Name:

α-​D-​Glucopyranoside, β-​D-​fructofuranosyl, diacetate hexakis(2-​methylpropanoate)

Constituent 2

Reference substance name:

Sucrose di(acetate) hexaisobutyrate

EC Number:

204-771-6

EC Name:

Sucrose di(acetate) hexaisobutyrate

Cas Number:

126-13-6

IUPAC Name:

Sucrose Acetate Isobutyrate

Details on test material:

Identification and Characterization
The test material, SAIB-SG, Lot No. 86/43, is a clear syrup. It was received at HLA in two shipments on August 13 and October 24, 1986, and was assigned HLA Sample No. 50707569. Before initiation of treatment and at 6-month intervals thereafter, samples of the test material were shipped to Eastman Chemical Division of Eastman Kodak Company for stability analysis. Information on the synthesis methods, purity, composition, and other
characteristics that define the test material are on file with the Sponsor.

Storage
The test material was stored at room temperature.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animal
Male and female CDF®(F-344)/CrlBR rats were obtained from the Kingston, New York, facility of the Charles River Laboratories, Inc. (Wilmington, Massachusetts). A prestudy colony health evaluation which included clinical and anatomical pathology was done on 10 4-week old and 10 10-week old rats per sex. In addition, a health monitoring report which included results from serology, bacteriology, parasitology, and gross anatomical pathology was provided by Charles River Biotechnical Services, Inc. The results are on file at HLA. Rats for the study were received on March 18, 1987, and were 6 weeks old and weighed between 79 and 130 g at initiation of treatment. Rats are frequently used in safety evaluation studies as a representative of a rodent species.

Identification
Upon arrival, each animal was assigned a temporary number. Before being placed on test, each animal was assigned a permanent number and identified with an ear tag. All data collected from an animal were recorded under one of these numbers.

Acclimation
The animals were received on March 18, 1987, and acclimated in Animal Room 345for 12 days before being placed on test. During acclimation, the animals wereexamined daily for abnormalities indicative of health problems. In addition, the following procedures were done: weekly body weights, food consumption for 1 week, clinical pathology determinations and necropsy on 10 animals/sex, and an ophthalmic examination. Twelve of 19 animals found with eye lesions were bled for a murine virus antibody determination for sialodacryoadenitis (SDA)/rat coronavirus, then sacrificed and discarded. All other animals not selected for the study were also sacrificed and discarded.

Housing and Maintenance
Animal Room 345 was used exclusively for this study. Environmental controls for the animal room were set to maintain a temperature of 72 deg F ± 3 deg, a relative . humidity of 50% ±20%, at least 10 changes/hour of filter, 100% outside air, and a 12-hour light/12-hour dark cycle. Variations from these conditions were documented and considered to have had no effect on the outcome of the study. The animals were housed individually. The cages were changed weekly and the racks were repositioned in the animal room weekly except during the last two weeks of the study. Animal husbandry and housing comply with the standards outlined in the "Guide for Care and Use of Laboratory Animals."

Teklad NIH07 Open Formula Rat and Mouse Diet was provided ad libitum, except when the animals were fasted. The lot numbers were recorded. Each lot of diet was analyzed for nutritional components and environmental contaminants before being used for the study. Results of the basal diet analyses are on file at HLA.

Water was provided ad libitum from an automatic system except when the health of an animal required that water be provided from a water bottle. Samples of the water are analyzed semiannually by HLA for total dissolved solids, conductivity, and specified microbiological content and for selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons. The results are on file at HLA. There were no known contaminants in the food or water that would have interfered with this study.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

acetone

Details on oral exposure:

Dose Administration
Because the route of human exposure to SAIB is oral, the test material was administered by dietary admixture for at least 52 weeks.

Dose Preparation
Fresh diets were prepared weekly. Dietary concentrations were based on the test material as supplied. The test diets were prepared by weighing the amount of NIH07 diet needed to prepare a dose level into a mixing bowl. The amount of test material needed to prepare that dose level was weighed into a tared beaker, and nanograde acetone (EM Science Company) was added. The amount of acetone added to all dose levels was equivalent to one-half the weight of SAIB used in the highest dose level for a given week. The SAIB in acetone was heated in a water bath at 70 deg C to dissolve the test material, then added to the food in the mixing bowl. The beaker containing the SAIB and acetone was cleaned using food from the mixing bowl, and the residual material was added to the mixing bowl. The test diet was then mixed for 30 minutes. This procedure was repeated for each dose level. Control diets were treated in the same manner with acetone alone. In order to achieve a constant dosage per kg of body weight, adjustments in test material concentrations were made using projected body weights and food consumptions. These adjustments were made weekly for the first 12 weeks of the study, then every fourth week thereafter. Before being fed to the animals, finished diets were stored at elevated room temperature {approximately 78 deg F) for at least 8 days to facilitate the evaporation of acetone.

Reserve samples from each mixed batch were retained in a freezer, then discarded after completion of the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Analyses
The Sponsor provided a verified method to assay for the test material in the basal diet (Appendix G). Minor method modifications are on file at HLA. The following analyses were done by HLA.

Homogeneity.
Before initiation of treatment, one sample each was taken from the top, bottom, and two opposing sides of the mixing bowl of the finished diet for three dietary concentrations (chosen to cover the anticipated dose range). Samples were assayed for the test material to verify that the mixing procedures resulted in homogeneous finished diets.

Stability
Samples were assayed periodically from several batches of diets to verify the stability of SAIB in diets. A preliminary study (Study A) was done using diets with 3,000, 25,000, or 50,000 ppm SAIB. The diets were mixed for 30 minutes. One set of samples was taken from each of the diets for SAIB analysis on the day of mixing (Day 0). Another set of samples was stored frozen and analyzed for SAIB on Day 44. On Day 0, the fresh diets were transferred to uncovered polypropylene containers and placed in a room kept at ambient temperature. The following day (Day 1), feed jars were filled from each mix and left in the same room overnight. On Days 2 and 9, samples were taken from the feed jars and analyzed for SAIB.

A second stability study (Study B) was done to assess the effect of elevated temperature on SAIB. In this study, diets containing 3,000 or 50,000 ppm SAIB were mixed for 30 minutes. Samples were taken for SAIB analyses on the day of mixing (Day 0) and diets, in uncovered polypropylene containers, were placed in a temperature controlled room with an elevated temperature (approximately 78 deg F). The diets were stirred once on Days 1 and 2 and then a portion of each diet was placed in feed jars. Samples were taken on Days 3 and 7 from the polypropylene
containers and analyzed for SAIB.

In a third study (Study C), the stability of SAIB was assessed in diets that were prepared for Week 3 of HLA Study No. 6914-101. On the day of mixing, (Day 0), the diets were placed in a temperature-controlled room with an elevated temperature (approximately 78.F). Samples were taken on Day 9, kept frozen for 3 days, and then analyzed for SAIB. On Day 9, a portion of each diet was placed in feed jars and kept at ambient temperature. Samples were taken on Day 19, kept frozen for 4 days, and then analyzed for SAIB. Because of the low recovery seen for the samples taken on Day 9, archive samples that had been frozen for 63 days were also analyzed for SAIB.

Verification of Dose Levels
To ensure that animals were exposed to the proper dose levels, all finished diets were assayed weekly for the first 12 weeks. Thereafter, one dose level was sampled each week (dose levels were selected sequentially), and stored in a freezer; these samples and a sample of the control diet were analyzed as a group approximately every 4 weeks.

Acetone Analyses
To determine the time required to achieve residual levels of less than 20 ppm acetone in the test diets, one set of samples for a selected dose level and control was assayed before study initiation for acetone periodically until this limit was reached for 2 consecutive days. In addition, all finished diets that were analyzed to verify dose levels of SAIB were also analyzed for acetone.

Duration of treatment / exposure:

Rats were fed diets containing SAIB for a minimum of 52 weeks.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
2.0 g/kg body weight x day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1.0 g/kg body weight x day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.5 g/kg body weight x day
Basis:
actual ingested

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

to the study using a computer-generated randomization. Individual body weights at the time of randomization did not exceed ±2 standard deviations from the mean body weight for each sex, and group mean body weights for each sex were not statistically different at the 5% level. A previous study of SAIB in the rat has demonstrated that this additive is well tolerated without toxicological changes at dietary concentrations up to 10% (Procter et al, 1971). It is generally accepted that dietary levels of non-nutrient substances should not exceed 5% in long-term rodent studies. This principle is set forth in numerous study guidelines. Accordingly, for this study, a maximum dose level on a body weight basis (2 g/kg} was selected that would be approximately equivalent to a 5% SAIB dietary concentration. The low and intermediate doses of SAIB selected were 0.5 and 1 g/kg.

Examinations

Observations and examinations performed and frequency:

Antemortem Observations
All animals were observed at least once in the morning and once in the afternoon for mortality, twice daily for moribundity, and signs of toxicity on all days except those listed in the protocol deviations (Appendix A). A physical examination was done once each week.

Ophthalmic Examinations
Ophthalmic examinations were done on all animals before the initiation of treatment and on all surviving animals during Weeks 26 and 52.

Body Weights
Individual body weights were recorded twice before initiation of treatment, at initiation of treatment, weekly thereafter, and on the day of necropsy.

Food Consumptions
Individual weekly food consumptions were recorded once before the initiation of treatment and every week thereafter.

Clinical Pathology
The hematology, clinical chemistry, and urinalysis tests listed below were done on all animals during Weeks 27 and 53. Hematology and clinical chemistry tests were also done on 10 animals/sex before initiation of treatment. Animals were fasted overnight; water ~as provided ad libitum. The animals were anesthetized with ketamine and blood was collected from the orbital plexus. For urinalysis tests, the animals were acclimated to metabolism cages for 24 hours, and then urine was collected in vials on ice for approximately 16 hours. The animals were fasted throughout the collection period; water was provided ad libitum.

HEMATOLOGY:
Red blood cell count
Hemoglobin
Hematocrit
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Reticulocyte count
Platelet count
Prothrombin time (during Week 53 only)
White blood cell count
Differential white blood cell count
(relative and absolute)
Nucleated red blood cell count
Corrected white blood cell count
Segmented neutrophil count
Band neutrophil count
Lymphocyte count
Monocyte count
Eosinophil count
Basophil count

CLINICAL CHEMISTRY:
Glucose
Total protein
Albumin
Globulin
Albumin/globulin ratio
Tot a 1 b i 1 i r·u b i n
Cholesterol
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Gamma glutamyl transferase
Urea nitrogen
Creatinine
Calcium
Inorganic phosphorus
Sodium
Potassium
Chloride
Electrophoresis (Albumin, Alpha-1 globulin, Alpha-2 globulin, Beta globulin,Gamma globulin)

URINALYSIS:
Specific gravity
Urine volume (16-hour)
Urine chemistry strip
Urine pH
Urine protein
Urine glucose
Urine ketone
Urine bilirubin
Urine blood
Urine urobilinogen
Urine appearance
Urine sediment (Red blood cells, White blood cells, Epithelial cells, Bacteria, Casts, Crystals)

LIVER FUNCTION TEST:
A bromsulphalein clearance test (SSP) was done on all animals in the control and 2-g/kg dose groups during Weeks 23 and 48. Animals were fasted overnight; water was provided ad libitum.

Sacrifice and pathology:

After 52 weeks of treatment, all surv1v1ng animals were fasted overnight, then anesthetized, weighed, exsanguinated via the vena cava, and necropsied. These procedures were also done for 10 animals/sex before initiation of treatment. Serum was collected from all animals necropsied after 52 weeks of treatment. The serum will be retained for possible future analysis.

All animals, regardless of fate, were necropsied. The necropsy included a macroscopic examination of the external surface of the body; all orifices; the cranial cavity; carcass; the external surfaces of the brain and spinal cord; the nasal cavity and paranasal sinuses; and the thoracic, abdominal, and pelvic cavities and viscera. The following tissues {when present) were preserved in 10% phosphate-buffered formalin {except eyes were preserved in Zenker's solution):

Adrenal glands*
Aorta
Bone marrow (femur)*
Brain (three sections)*
Cecum*
Cervix
Colon*
Common bile duct*
Duodenum*
Epididymides*
Esophagus*
Eyes*
Heart*
Ileum*
Jejunum
Kidneys*
Larynx
Lesions*
Liver (three sections)*
Lungs (with mainstem bronchi)*
Lymph nodes (cervical, thoracic,
and mesenteric)*
Muscle {skeletal, posterior to
femur to include sciatic nerve)*
Ovaries and fallopian tube*
Pancreas*
Pituitary (preserved in situ)*
Nasal turbinates
Prostate*
Rectum
Rib*
Salivary glands*
Seminal vesicles*
Skin with mammary gland*
Skull
Spinal column (thoraco-lumbar)*
Spleen*
Sternum*
Stomach*
Testes*
Thymus*
Thyroid gland (with parathyroid)*
Tongue
Trachea*
Urinary bladder*
Uterus*
Vagina
Zymbal's gland*

* Examined histologically from all control and high-dose animals and all animals that died or were sacrificed in a moribund condition; kidneys,
liver, lungs, and all lesions were also ex~mined microscopically from all low- and mid-dose animals. Liver sections were taken from animals at the terminal sacrifice and processed for electron microscopy (described in Attachment A to Amendment No. 2) and then shipped to Experimental Pathology Laboratories, Inc. (Herndon, Virginia) for analyses. The results of electron microscopy are in Appendix G. Additional sections of liver from all animals at the terminal sacrifice were collected, homogenized, and retained below -70"C for possible future analysis for palmitoyl CoA oxidase; the latter analyses were not done. At the terminal necropsy, in addition to terminal body weights, the following organs (when present) were weighed (paired organs were weighed separately):

Brain
Heart
Kidneys
Liver
Ovaries
Testes
Organ-to-body weight percentages and organ-to-brain weight ratios were calculated. Bone marrow smears were prepared from all animals at the terminal necropsy, stained with Wright's stain, and retained for possible future examination. Tissues to be examined histologically were embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

Statistics:

Levene's test is done to test for variance homogeneity. In the case of heterogeneity of variance at p ~ 0.05, the following transformations are used to stabilize the variance.

Log X = Data analyzed following log 10 transformation
X2 = Data analyzed following square transformation
X112 Data analyzed following square root transformation
1/X Data analyzed following reciprocal transformation
Arcsine X112 = Data analyzed following angular transformation
Rank X = Data analyzed following rank transformation

Analysis of variance (ANOVA) is done on the homogeneous or ranked data. If the ANOVA is significant, Dunnett's t-test is used for pairwise comparisons between groups. When no transformation establishes variance homogeneity at p < 0.001, the data are also examined by nonparametric techniques. These statistics include the Kruskal-Wallis H-test and, if this test is significant, the Nemenyi-Kruskal-Wallis test for multiple comparisons6 or the Wilcoxon-MannWhitney two-sample rank test. Standard one-way ANOVA was used to analyze body weights; body weight gains; cumulative body weight gains; food consumptions; clinical chemistry and hematology values (except erythrocyte morphology); urine pH, volume, and
specific gravity values; organ weights; organ-to-body weight percentages; and organ-to-brain weight ratios. Group comparisons found to be statistically significant at the 5.0% two-tailed probability level are indicated with an asterisk (*).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

.

Mortality:

mortality observed, treatment-related

Description (incidence):

.

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

Acclimation
From a supply of 110 males and 108 females, 10 animals/sex were removed for baseline clinical pathology tests, 19 animals were excluded from consideration for the study because of eye lesions observed at the baseline ophthalmic examination, and 11 were excluded because their body weights were not within the randomization weight limits (i.e., ±2 standard deviations from the mean of each sex). The results of the SDA/rat coronavirus test done on 12 animals with eye lesions were negative. The animals were released from acclimation by the laboratory animal veterinarian on March 27, 1987.

Dose Analyses
The actual concentrations of SAIB in the diets ranged from 89% to 106% of claim. Dietary acetone levels were below the limit of detection (i.e., 20 ppm) for Weeks 1 through 30 except one Week 5 diet which had assay results of 23.9 and 22.5 ppm. Dietary acetone levels ranged from below the limit of detection to 70.3 ppm for Weeks 31 through 53.

Antemortem Observations
There were no test material-related antemortem observations. There was 1 male given 2 g/kg (Animal No. C56928) and 1 female given 1 g/kg
(Animal No. C56894) that died during the study and 1 female given 0.5 g/kg (Animal No. C56860) and 1 female given 2 g/kg (Animal No. C56936) that were sacrificed in a moribund condition. Animal No. C56928 died and Animal No. C56894 was sacrificed after BSP blood collection. There was also 1 control male (Animal No. C56797) that died during the Week 26 clinical pathology bleeding due to an accidental overdose of anesthetic. None of these deaths appeared to be related to treatment with the test material.

Ophthalmic Examinations
There were no test material-related ophthalmic observations.

Body Weights
Group mean body weights were lower for males and females given 2 g/kg SAIB. These differences were statistically significant for females at Weeks 19, 20, 26 through 47, and 50 through 52. There were no statistically significant differences for males given 2 g/kg. lower body weights at Weeks 23 and 48 for males and females given 0 or 2 g/kg were a result of the animals being fasted for BSP tests.

Body Weight Gains
Cumulative body weight gains for females given 2 g/kg SAIB were significantly lower for Weeks 19 through 23 and 25 through 52. Cumulative gains for males given 2 g/kg SAIB were also lower throughout the study, but significant differences occurred only for Weeks 13 and 51.

Food Consumptions
Group mean food consumptions were slightly lower throughout the study for females given 2 g/kg SAIB. There were no consistent differences in food consumptions for males.

SAIB Consumptions
Overall mean SAIB consumptions ranged from 94% to 97% of the theoretical dose.

Clinical Pathology
There were no test material-related changes in clinical pathology parameters.

Liver Function Test
There were no significant differences for BSP clearance between the groups.

Anatomical Pathology
Terminal body weights were significantly lower in females given 2 g/kg. This difference was treatment related. Organ-to-body weight percentages for brain and heart were significantly higher for females given 2 g/kg. These differences were due to lower body weights in this group. There were no macroscopic or microscopic changes associated with any of the organ or body weight differences, and there were no other treatment-related macroscopic changes. Hepatocellular adenoma, a benign, well-differentiated, circumscribed neoplasm of hepatic origin, was found in two females given 2 g/kg. This is a relatively common spontaneous tumor in rats; the occurrence of these tumors in this group were considered coincidental and not related to treatment.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

2

Key result

Dose descriptor:

NOAEL

Effect level:

> 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the no-observable-effect level for SAIB when administered to rats in their diet for 52 weeks is 1 g/kg body weight/day for females and greater than 2 g/kg body weight/day for males.

Executive summary:

This study was conducted to assess the chronic toxicity of sucrose acetate isobutyrate (SAIB) when administered to rats in their diet for at least 52 weeks. Twenty animals/sex/group were given 0, 0.5, 1, or 2 g SAIB/kg body weight/day (g/kg) in the basal diet for 52 weeks. Physical examinations and body weight and food consumption measurements were done weekly. Ophthalmic examinations were done before the initiation of treatment and during Weeks 26 and 52. Hematology and clinical chemistry tests were done on 10 animals/sex before initiation of treatment and hematology, clinical chemistry, and urinalysis tests were done on all surviving animals during Weeks 27 and 53. A bromsulphalein liver clearance test was done on all surviving animals in the control and 2-g/kg dose groups during Weeks 23 and 48. Necropsies were done on the 10 animals/sex bled for clinical pathology tests before initiation of treatment, on all animals that died or were sacrificed in a moribund condition, and on all surviving animals during Week 53. Tissues were collected and preserved from all animals and examined microscopically from animals in the control and 2-g/kg dose groups and all animals that died or were sacrificed in a moribund condition. Kidneys, liver, lungs, and all macroscopic lesions collected were examined microscopically from all animals. There were no test material-related antemortem observations, effects on clinical pathology variables or the bromsulphalein clearance test, or macroscopic or microscopic anatomical pathology changes. Body weights, cumulative body weight gains, and food consumptions for females given 2 g/kg SAIB were lower than those of the controls throughout the study and body weights for females given 0.5, 1, or 2 g/kg were 6%, 10%, and 7% lower, respectively, than those of the control group at Week 52. Higher organ-to-body weight percentages for the brain and heart corresponding to lower terminal body weights were also seen for this group. Males given 2 g/kg also had slightly lower body weights and cumulative body weight gains. Based on the results of this study, the no-observable-effect level for SAIB when administered to rats in their diets for 52 weeks is 1 g/kg body weight/day for females and greater than 2 g/kg body weight/day for males.