Registration Dossier

Administrative data

Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
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 guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
no guideline followed
Principles of method if other than guideline:
3-generation reproductive and teratology study in rats
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
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:
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. It was stored in plastic containers at room temperature. Before initiation of treatment and at 6-month intervals thereafter, samples of the test material were taken and shipped to Eastman Chemicals Division of Eastman Kodak Company for stability analysis. Information on the methods of synthesis, purity, composition, or other characteristics that define the test material is on file with the Sponsor. Nanograde acetone was used as the vehicle. Stability, purity, and composition are on file with the manufacturer. Lot numbers are on file in the raw data. A reserve sample of each of the test and vehicle (acetone) materials was taken before initiation of treatment and retained. These samples will be disposed of as authorized by the Sponsor, upon acceptance of the final report. The test material sample will be retained in accordance with 21 CFR 58.195. All remaining test material was transferred to the Sponsor's designee. The test material was stored at room temperature. The acetone 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:
Albino male and female Fischer 344 [CDF®(F-344)/CrlBR] rats (guaranteed nonlittermates), purchased as weanlings from the Portage, Michigan, facility of Charles River Laboratories, Inc. (Wilmington, Massachusetts), were used as the FO generation. Rats are sensitive to a number of agents that interfere with reproduction and are routinely used to determine the potential of chemicals for producing adverse effects on the reproductive system. In addition, reproductive toxicity studies in rats are required by the various regulatory agencies. Fischer 344 rats are currently being used for toxicity studies by the U.S. National Toxicology Program.

Identification
A temporary number was assigned to each FO animal upon arrival and to each F1 pup selected to be an adult. Before being placed on test, each animal
selected ·;or the reproduction study or necropsy was assigned a permanent identification number and was identified with a numbered metal ear tag. All data collected from an animal were recorded and filed under one of these numbers. Color-coded cage identification cards were placed on all cages
specifying the study number, study director, animal number, sex, test material, dosage, and study initiation date.

Acclimation
An initial shipment of 132 males was received on October 8, 1986, and taken to Animal Room No. 331. Because initiation of the study was delayed, the animals were removed from the study on October 16, 1986. A second shipment of males was received on November 26, 1986, and taken to Animal Room No. 331. Females were received on January 21, 1987, and taken to Animal Room No. 329. During acclimation, the animals were examined for clinical abnormalities indicative of health problems (e.g., diarrhea, ectoparasites, rough hair coat, nasal or ocular discharge, evidence of injury, etc.), body weights were recorded for 1 week during acclimation. Males were released from acclimation on December 8. 1986. and placed on test on December 8, 1986. Females were released from acclimation on January 30, 1987, moved to Animal Room No. 331 on that date, and placed on test on February 2, 1987.

Housing and Maintenance
Animal Room 331 was used exclusively for this study. Because the females were received approximately 8 weeks later than the males, they were initially kept in Room No. 329 and were moved to Room No. 331 after being ·released from acclimation. Environmental controls for the animal room were set to maintain a temperature of 72 deg. F ±3·, a relative humidity of 50% ±20%, at least 10 changes/hour of filtered, 100% outside air and a 12-hour light/12-hour dark cycle. Variations from these environmental conditions were documented and were not considered to have had any effect on the outcome of the study.

Animal husbandry and housing were in compliance with the standards outlined in the "Guide for the Care and Use of Laboratory Animals. "1 Care was taken to ensure that animals were not disturbed unnecessarily. During the growth and holding phases of the study, animals were housed individually in suspended, stainless steel, screen-bottom cages held on racks, with animal cage boards lining the urine- and feces-collecting pans. During breeding periods, doublesized stainless steel cages were used to house one male and one female rat each. Cage boards were changed at least twice weekly, and animals were transferred to clean cages at least biweekly except during the breeding period. Pregnant rats (approximately Day 20 of gestation), rats with young, and females that did not show evidence of positive mating were housed in polycarbonate cages fitted with water bottles filled with tap water. A bedding material consisting of heattreated hardwood chips covered the bottom of the polycarbonate cages and was
changed at least once weekly. Racks were repositioned within the animal room periodically to minimize the effects of environmental variations; however, rack position was not documented. Variations from these procedures were documented and included in the Protocol Deviations. A ground form of Nlh07 Open Formula Rat and Mouse Diet was provided ad libitur. The lot numbers werE recorded. The diet was analyzed by HLA for nutrients and environmental contaminants. Deviations from the specified limits were documented.

Water was provided ad libitum. Samples of the water are analyzed 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 with HLA. There were no known contaminants in the food or water that would have interfered with the study.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
acetone
Details on exposure:
Test Material Administration
Because humans are exposed to the test material by the oral route as an intentional food additive, it was administered to the animals in the basal diet.

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 amount of SAIB used in the highest dose level for a given week. The SAIB in acetone was heated in a water bath at 70"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.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.
Details on mating procedure:
Breeding of the FO adults for Fl litters, Fl adults for F2a litters, F1 adults for F2b litters, and F2a adults for F3 litters was initiated on February 16, 1987, June 29, 1987, September 21, 1987, and December 1, 1987, respectively, after test diets had been fed to the animals for at least 10 weeks. Breeding of the F1 adults for F2b litters was initiated 2 weeks after completion of weaning of the F2a litters. In each generation, oreeding was initiated by selecting one male and one female at random from animals on the same treatment and placing each pair of animals in a separate double-sized, screen-bottom cage. Assignment of pairs was done using computer-generated random number tables. The first female in the randomization sequence was placed ~~ith the first male in the randomization sequence from the same group. The next female was placed with the next male, and this sequence was repeated until all available animals were paired. Sibling and half-sibling matings were avoided. Each pair had a maximum of 21 days to achieve mating.
Once mating had occurred, the males and females were separated. Females that showed no evidence of mating were placed in nesting boxes after the mating period. Date mated, date positive sperm or vaginal plug was observed, and male mate number were recorded.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The Sponsor provided a verified method to assay the test material in the basal diet. Minor method modifications are on file at HLA. The
following analyses were done by HLA.

top, bottom, and two opposing sides of the mixing bowls of the finished diet of 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 form 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 SAI3 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 the diets, in uncovered polypropylene containers, were placed in a temperature-controlled room with an elevated temperature (approximately78 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. 6194-101. 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 fi~ished diets were assayed weekly for the first 12 weeks. Thereafter, one dose level was sampled each week (dose levels were selected sequentially), stored in a freezer, and analyzed every 4 weeks.

Acetone Analyses.
To determine residual levels of acetone in the test diets, all finished diets that were analyzed to verify dose levels of SAIB were also analyzed for acetone. The limit of detection for the assay was 20 ppm.
Duration of treatment / exposure:
The FO males and females received the test material at the appropriate dose levels in the diet continuously from study initiation (December 8, 1986, for males and February 2, 1987, for females) for 10 and 2 weeks, respectively, before mating; throughout mating, gestation, and lactation; and until necropsy on April 20 and 21, 1987 (males), and April 23 and 24, 1987 (females). The F1 males and females were exposed to the test material in utero; while nursing; continuously from weaning to test diets until mating (at least 10 weeks); throughout mating, gestation, and lactation for F2a litters and
through Day 20 of gestation for F2b litters; and until necropsy on November 9, 1987, for the males and on Day 20 of the F2b gestation (October 12 through 24, 1987) for the females. The F2a males and femctles were exposed to the test material in utero, while nursing, continuously from weaning to test diets until mating (at least 10 weeks), and throughout mating. Males were sacrificed and discarded upon completion of the breeding period (December 22, 1987). Females received the test material until sacrificed on presumed Day 14 of F3 gestation (December 16, 1987, to January 5, 1988).
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.5 g/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1.0 g/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
2.0 g/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
30
Control animals:
yes, sham-exposed
Details on study design:
Animals were assigned to the study using a computer-generated random numbers table. These were tre FO animals of the FO generation.At randomization, the weight variation of animals of each sex to be used did not exceed ±2 standard deviations of the mean weight, and the mean body weights for each group of each sex were not significantly different. The FO animals were mated to produce Fl litters.

Thirty males and 30 females from the Fl litters were selected from each group using a computer-gener·ated random numbers table and were continued on the treatment of their par·ents. These were the Fl animals of the Fl generation.The F1 animals were mated to produce F2a and F2b litters. The F2b litters were used for teratologic evaluation.

Thirty males and 30 females from the F2a litters were selected from each group using a computer-generated random numbers table and were continued on the treatment of their parents. These were the F2a animals of the F2a generation.The F2a animals were mated to produce F3 litters. The study was completed with the necropsy of the F2a females on Day 14 of the F3 gestation.

Examinations

Parental animals: Observations and examinations:
Vaainal Examinations. Vaginal smears were taken daily, and the presence of a copulatory plug or sperm in the vaginal smear was considered evidence of positive mating. The day on which such evidence was found was considered Day 0 of gestation, and the female was removed and housed individually. On Day 18 of gestation, the mated female was placed in a plastic cage.
Litter observations:
Litter Observations (F1 and F2a litters)

Birth (Day 0 of lactation).
As soon as possible after birth, the sex of each pup was determined, and the litter size (total number of pups born live andfound dead) was recorded. Each live pup was examined for external abnormalities, and the pups were weighed by sex by litter. Dead pups were examined grossly for cervical, thoracic, and abdominal visceral abnormalities. Following examination, dead pups were to be preserved in fixative; however, in some cases the pups were discarded inadvertently.

Day 4
The sex of each pup was determined, and the litter size (number of live pups) was recorded. The pups were examined for external abnormalities and
weighed by sex by litter after culling. litters with more than 10 pups were culled at random to produce, as nearly as possible, litters that contained five
males and five females. Culled pups were examined for cervical, thoracic, or abdominal visceral abnormalities and then sacrificed and discarded.

Day 28
The sex of each pup was determined, and the litter size (number of live pups) was recorded. Each pup was examined for external abnormalities and weighed. On Day 28 of lactation, or as soon as possible thereafter, one male study. For litters without a male or female pup, one weanling was selected at random from the pups of the available sex. When sufficient pups or litters were not available, male and female pups were selected at random from the remaining litters within groups to provide 30 male and 30 female weanlings/group. Records were maintained of the derivation of each pup so that sibling matings could be avoided. All pups not selected as future parents were sacrificed and discarded.

Cesarean Section and Fetal Observations (F2b and F3 Litters)
The Fl dams were sacrificed on presumed Day 20 of the F2b gestation and examined macroscopically. These animals were also examined for the number and distribution of fetuses in each uterine horn, the number of fetuses undergoing resorption, and the nLmber of corpora lutea. Live fetuses were removed from the uterus, sexed, examined for gross abnormalities, and weighed. Approximately one-half of the pups of each litter were preserved in Bouin's solution and subsequently examined for soft tissue abnormalities using a modification of Wilson's serial slicing technique (slices were discarded after the examination was completed); the remaining animals were placed in alcohol and then macerated in potassium hydroxide, stained with alizarin red, and examined for skeletal abnormalities. The F2b dams were sacrificed on presumed Day 14 of the F3 gestation. These animals were examined for the number of corpora lutea and implantations and discarded without further examination.
Statistics:
Standard one-way analysis of variance {ANOVA) was used,to analyze the following data for each sex: body weight; body weight gain; food consumption; days to mate; length of gestation; pup viability, body weights, and sex ratios; litter size (alive and dead); the number of corpora lutea and implantations; implantation efficiency; gravid uterine weight; and the number and percentage of live, dead, and resorbed (early and late) fetuses.
The statistical methods that are used ire diagrammed in Figure 1 and described below. Levene's test was done before ANOVA to test for variance homogeneity. In the case of hetero9eneity of variance at p < 0.05, the following transformations were used to stabilize the variance:
o Log X = Data analyzed following log e10 transformation
0 X2 = Data analyzed following square transformation
o X e 1/2 = Data analyzed following square root transformation
o 1/X = Data analyzed following reciprocal transformation
o Arcsine X e 1/2 = Data analyzed following angular transformation
o Rank X = Data analyzed following rank transformation

The ANOVA was then performed on the homogeneous or ranked data. If the ANOVA was significant, Dunnett's t-test was used for pairwise comparisons between groups. When no transformation establishes variance homogeneity at p < 0.001, the data were also examined by nonparametric techniques. These statistics include the Kruskal-Wallis H-test and, if this test was significant, the Nemenyi-Kruskal- Wallis test for multiple comparisons or the Wilcoxon-MannWhitney two-sample rank test. Reproduction indices were analyzed by the Cochran-Armitage test for trend and Fisher-Irwin exact test for heterogeneity. In addition, fetal viability of the F2b litters was evaluated by Student's t-test. All differences cited (higher/greater and lower/less} were based on comparisons with the control group. Group comparisons found to be statistically significant at the 5.0% two-tailed probability level are indicated with an asterisk.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BW were decreased 3-6% during first half of lactation at 1.0 and 2.0 g/kg females
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
BW were decreased 3-6% during first half of lactation at 1.0 and 2.0 g/kg females
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: no effects observed

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BW were reduced in F1 females on day 21 of lactation in 0.5 and 2.0 g/kg groups
Food consumption and compound intake (if feeding study):
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
food consumption and compound intake
Remarks on result:
other: no consistent statistically significant differences in body weight gains although slightly lower food consumption was noted .

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
> 2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: No effects observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, when SAIB was administered to rats continuously in the diet for three successive generations, the highest dose
level (2.0 g/kg) was considered to be the no-observable-effect level.
Executive summary:

Groups of 30 male and 30 female immature rats (FO animals) were fed diets that contained 0, 0.5, 1.0, or 2.0 g of sucrose acetate isobutyrate (SAIB)/kg of body weight (g/kg). Males and females received the test diets for 10 and 2 weeks, respectively. In addition, females received test diets continuously throughout mating, gestation, and lactation, until necropsy. The FO animals were then mated to produce F1 litters. Groups of 30 male and 30 female F1 animals were fed diets that contained 0, 0.5, 1.0, or 2.0 g of SAIB/kg for at least 10 weeks postweaning, and the breeding program was repeated to produce F2a animals. After weaning of the F2a litters, the Fl adults were remated to produce F2b litters, which were used for teratologic evaluation. The F2a animals (30/sex/group) were fed test diets containing 0, 0.5, 1.0, or 2.0 g of SAIB/kg for at least 10 weeks postweaning and then mated to produce F3 litters. Adult F2a males were sacrificed and discarded after the completion of mating. The study was completed when the adult F2a females were sacrificed on Day 14 of the F3 gestation, and the uterus was examined for the number of implantations. Antemortem data (i.e., antemortem observations, body weights, and food consumptions), reproduction data, and litter data were recorded. Test diets were fed continuously throughout the study. All FO and Fl adults were examined macroscopically, and reproductive organs and gross lesions were co:lected and preserved for possible examination. Tissues "masses" found in the peritoneal cavity were examined microscopically. The results of the study are summarized as follows:

  • Survival was 100% for males and females in all groups in the FO, Fl, and F2a generations.
  • There were no treatment-related antemortem observations.
  • Body weights were slightly (3% to 6%), but significantly, lower only during the first half of lactation at 1.0 and 2.0 g/kg for FO females and on Day 21 of lactation at 0.5 and 2.0 g/kg for F1 females.
  • There were no consistent statistically significant differences in body weight gains.
  • Food consumptions for F2a males and females were significantly lower during the premating period.
  • There was a significant trend in decreased fertility for F1 females during mating for the F2a litters; however, there were no statistically significant differences between the control and treated groups and no effect of continued SAIB treatment on subsequent matings (i.e., for F2b or F3 litters). The single observation of reduced fertility is not considered to be test material-related.
  • Fetal viability for F2b litters was significantly decreased at 2.0 g/kg based on analysis of variance, however, not when the values were compared by Student's t-test. This observation is not considered to be test material-related.
  • There were no treatment-related effects on fetal development through the period of organogenesis (i.e., Days 6 through 15 of gestation) based on teratologic evaluation of the F2b liiters.
  • There were no treatment-related macroscopic or microscopic changes.