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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
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:
1988
Report Date:
1988

Materials and methods

Principles of method if other than guideline:
The objective of this study was to determine the teratogenic potential of the test article in rabbits, in accordance with 1982 FDA Safety Assessment Guidelines for Direct Food and Color Additives.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
The test article was received from Eastman Chemical Products, Inc., Kingsport, Tennessee on August 15, 1986. The label on the container read as follows:

Read all cautionary information
FOR MANUFACTURING USE ONLY
EASTMANGD SAIB
sucrose acetate isobutyrate Special
Lot #86-43

The test material was described as a pale yellow gel ( 100, one-qt containers received)

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Eighty untreated, sexually mature four month old, v1rg1n female New Zealand White SPF rabbits were received from Hazleton Research Animals, Denver, Pennsylvania, on May 13, 1987. Upon receipt, the animals were assigned temporary animal numbers and housed individually in suspended stainless steel cages with "deotized" animal cage boards (D.A.C.B.) to control excretory odor. During the 26-day acclimation period, the animals were carefully observed for changes in appearance and behavior. From acquisition until sacrifice, each animal was provided with basal laboratory diet of Purinae Certified Rabbit Chow #5322 and tap water. Upon receipt, the rabbits were maintained on a measured dietary regimen for approximately two weeks after which the diet was available ad libitum. Tap water was freely available at all times. Throughout the study, all animals were housed in an environmentally controlled room. Temperature ranged between 64°F and 67°F, with a mean temperature +/- standard deviation of 66 +/- 0.9°F. Temperature was above protocol specifications eight times and was inadvertently not recorded once. In the opinion of the Study Director, this did not affect
the integrity of the study. Humidity ranged between SO% and 60% with a mean humidity +/- standard deviation of 54 +/- 4.0%. Fluorescent lighting
provided illumination 12 hours per day. Does were housed individually in suspended stainless steel cages from receipt until sacrifice. Nesting
material was not provided s1nce the females were sacrificed prior to delivery. Each female was identified by cage, group and individually by
a Monel® metal ear tag bearing its animal number. The individual animal number plus the IRDC study number composed a unique identification number for each animal. The females were approximately 5 months old at the time of insemination and weighed between 2774 and 3736 g on gestation day 0. Approximately three weeks prior to insemination the does were superovulated by an injection of 50 U.S.P. units of human chorionic gonadotropin (lot #86 D 057, received from Cooper Drugs) via a marginal ear vein.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The selected route of administration was oral since this was considered the most closely equivalent to the typical human exposure. Dose levels were selected on the basis of previously conducted range-finding studies in which it was determined that the test article, SAIB, could be administered to New Zealand White rabbits orally by gavage 1n a corn oil vehicle at dosage levels of 1,200 mg/kg/day or less given the limitation of the tolerated maxtmum dosage volume for corn oil of 1 ml/kg, 2X/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to test material preparation, the test article, SAIB, was liquefied by heating to approximately 37 degrees C in a water bath. The appropriate amount of SAIB for each group was weighed into a graduated cylinder. The vehicle, corn oil (warmed to approximately 37 degrees C) was added to the graduated cylinder and the cylinder was shaken by hand. After this mixture was allowed to cool to room temperature, a sufficient quantity of vehicle (at room temperature) was added to the cylinder to yield 2,000 ml of prepared test material. The flask was again shaken by hand and heated, if necessary, to achieve a homogeneous mixture. The contents of the flask were divided into daily aliquots (2 per day). The test material was prepared once for the study and stored under refrigeration until the day prior to use. The prepared test material was analyzed at IRDC prior to administration and on the last day of administration.
Details on mating procedure:
Insemination Procedure
Six proven male rabbits of the same breed and source were selected to serve as donors. Semen was collected using an artificial vagina and the gelatinous plug was removed from the ejaculate. The semen was immediately evaluated for motility and was used for insemination only if the motility was 60% or greater as determined by the technician. The ejaculate was diluted with 3.0 ml of 0.9% Sodium Chloride for Injection, U.S.P., at 33-37°C and 0.3 ml of this dilute semen was introduced into the anterior vagina of the female using an insemination pipette. A minimum of 0.5 million sperm/doe was provided at insemination. Immediately after insemination, ovulation was induced by an injection of 100 U.S.P. units of human chorionic gonadotropin (lot #86 D 057, received from Cooper Drugs) into a marginal ear vein. Semen from one male was used to inseminate an equal number of females in each group. Insemination procedures were performed on three consecutive days and an equal number of females were inseminated in each group per day. The day of insemination was designated as day 0 of gestation.
Duration of treatment / exposure:
Test material was administered on gestation days 7 through 19.
Frequency of treatment:
Prior to daily administration, daily aliquots of prepared test material were warmed (approximately 40 degrees C) tn a water bath. The test material was prepared at concentrations (based on density of 1112.3 mg/ml) to permit twice daily (b.i.d.) administration of dose levels of 250, 425 and 600 mg/kg/dose at volumes of 0.685, 0.84, and 1.0 ml/kg, respectively which yielded total daily doses of 500, 850 and 1,200 mg/kg/day at total dose volumes of 1.37, 1.68, and 2.0 ml/kg, respectively. The test article was administered twice daily (b.i.d.) on days 7 through 19 of gestation. Daily doses were approximately 6 hours apart. Test article administration was accomplished by intragastric intubation using 3 or 5 cc disposable plastic syringes and 12 gauge, 15 cm long curved stainless steel dosing needles. Dosing began approximately 1 1/4 to 4 hours (first dose) or 7 1/4 to 10 hours (second dose) after initiation of the illuminated phase of the photoperiod. The control group received the vehicle only on a comparable regimen at a total daily volume of 0.92 ml/kg. Individual dosages were specified to be determined from individual body weights recorded on gestation day 0, but the most recently recorded individual body weights were used. This deviation is not considered by the Study Director to have affected the outcome of the study in any way. The prepared test material was analyzed at IRDC prior to administration and on the last day of administration.
Control animals:
yes, concurrent vehicle
Details on study design:
At the end of the acclimation period, all animals were weighed and subjected to a detailed physical examination. Animals considered suitable for study were randomly assigned to one control group and three treatment groups of 16 rabbits each by the following computer-generated system. Animal numbers and corresponding body weights were entered onto magnetic tape which was used as the data source for the randomization procedure. The mean body weight and standard deviation were calculated and a computer-generated edit developed a listing of those animals whose body weights were within +/- 1.5 standard deviations of the mean. From the qualifying animals, the randomization procedure selected and assigned the required number of animals. Bartlett's test for homogeneity of variance was applied to the groups; the groups were judged to be homogeneous.

Examinations

Maternal examinations:
Survival, Appearance and Behavior
Throughout the study the females were observed twice daily for mortality and overt changes in appearance and behavior. The presence and duration of clinical signs of toxicity were recorded once daily during gestation days 7 through 19, 20, or 21 and on gestation day 29. Females not surviving to the scheduled sacrifice were necropsied in an attempt to determine the cause of death. The fetuses from these does were externally examined and preserved in neutral buffered 10% formalin for possible future evaluation. Maternal tissues were preserved in neutral buffered 10% formalin for microscopic examination as deemed necessary by the gross findings.

Body Weights
Individual maternal body weights were recorded on gestation days 0, 7, 13, 20 and 29.

Food Consumption
Individual maternal food consumption was recorded on gestation days 0, 7, 13, 20 and 29.

Cesarean Section Observations
On gestation day 29, all surviving females were sacrificed by an injection of sodium pentobarbital via a marginal ear vein. Immediately following sacrifice, the uterus and ovaries were exposed by an abdominal incision. The number and location of viable and nonviable fetuses, early and late resorptions and the number of total implantations and corpora lutea were recorded. The uterus was then excised and the fetuses removed. The abdominal and thoracic cavities and organs of the does were examined for grossly evident morphological changes and the carcasses discarded. Maternal tissues were preserved in neutral buffered 10% formalin for histopathological examination as deemed necessary by gross findings. Uteri from females that appeared nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantations.
Fetal examinations:
All fetuses were individually weighed, tagged and examined for external malformations and variations. Each fetus was dissected, internally sexed and examined for visceral malformations and variations, including the brain by a mid-coronal slice. The heart was dissected by a modification of the method described by Staples. The eviscerated skinned fetuses were fixed in alcohol, macerated in potassium hydroxide, stained with Alizarin Red S and cleared with glycerin by a method similar to that described by Dawson for subsequent skeletal examination. All gross, visceral, and skeletal alterations observed in this study have been placed into one of two categories, malformations or developmental variations. These are defined as follows: Malformations are those structural anomalies that alter general body conformity, disrupt or interfere with body function, or are generally thought to be incompatible with life. Specific examples of processes that result in maldevelopment include marked/severe misshapening, asymmetry or irregularity of structure brought about by fusion, splitting, disarticulation, malalignment, hiatus, enlargement, lengthening, thickening, thinning, or branching. Absence (agenesia) of parts or whole structures is also considered a malformative process. Developmental variations are those alterations in anatomic struc:ture that are considered to have no significant biological effect on animal health or body conformity, representing slight deviations from normal. Most examples of alterations placed in the variant category are minor variations in size and form of normally present ossification centers. While these are evaluated on a precise day of development, some variation is expected related to when conception and implantation actually occurred. Thus, differences in the pattern of ossification, manifested either as retardation or as acceleration of apparent osteogenesis, are common findings. Also included in this category are slight misshapening or misalignment of structures, processes involving continued development (bilateral skeletal centers not yet fused, incomplete maturation of renal papillae, presence of vestigial structures, etc.), and development of extra ossification sites. Several clarifications are necessary with respect to tabulation of these categories as follows: Tabulations refer to numbers of fetuses/litters affected. When a malformation exists in a particular site, a variation in the same site is also tabulated, e.g. fused ribs/13th rudimentary rib. In cases where two or more variations of the same type or structure exist in the same specimen, all are counted with a single exception: a unilateral rudimentary rib is not tabulated when an extra full rib is present. The kidney was evaluated according to criteria described by Woo and Hoar and only grade 0 lesions of the renal pelvis and papilla (absent papillae) were tabulated as variations. Dilatation of the ureter was also included in the tabulation, whether or not it accompanied renal lesions.
Statistics:
All statistical analyses compared the treatment groups with the control group, with the levels of significance at p<0.05 and p<0.01. All means were accompanied by standard deviations. Male to female fetal sex ratios were compared using the Chi-square test criterion with Yates' correction for 2 x 2 contingency tables and the proportions of litters with malformations and variations were compared usirtg Fisher's exact probability test as described by Siegel to determine the significance of differences. The proportions of resorbed and dead fetuses and pre- and postimplantation losses were compared by the Mann-Whitney U-test as described by Siegel and Weil to determine the significance of differences. Mean numbers of corpora lutea, total implantations and live fetuses and mean maternal and fetal body weights and maternal body weight changes were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variance and the appropriate t-test (for equal or unequal variance) as described by Steel and Torrie using Dunnett's multiple comparison tables to determine the significance of differences.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

+

Applicant's summary and conclusion

Conclusions:
Administration of SAIB at twice daily doses of up to 600 mg/kg to rabbits during gestation day 7 through 19 resulted in no meaningful differences in maternal antemortem and necropsy observation, body weight gain, food consumption or Cesarean section observations relative to those of the control group were seen. No evidence of an adverse effect on fetal morphology was seen at any tested level. Data indicate that the 1,200 mg/kg/day level was determined to be the "no observable effect level" (NOEL) with respect to developmental toxicity, including teratogenesis, in this study.
Executive summary:

Inseminated female New Zealand White SPF rabbits randomly assigned to one control and three treatment groups of 16 animals each were used to determine the teratogenic potential of Sucrose Acetate Isobutyrate (SAIB). Dosage levels of 250, 425 and 600 mg/kg body weight/dose at volumes of SAIB of 0.685, 0.84, and 1.0 ml/kg body weight, respectively, were administered orally by gavage twice daily on days 7 through 19 of gestation to yield total daily doses of 500, 850 and 1,200 mg/kg body weight/day at total daily volumes of 1.37 1.68, and 2.00 ml/kg body weight, respectively (contained in a volume of 0.92 ml/kg body weight corn oil). The control group received the vehicle only, corn oil, on a comparable regimen at a total daily volume of 0.92 ml/kg. Cesarean sections were performed on all surviving females on gestation day 29 and the fetuses were removed for teratologic evaluation. Two high dose animals died on gestation day 17. One death was determined to be due to mucoid enteritis and the other was from unknown cause(s). The remaining animals on study survived to scheduled sacrifice. There was a slight increase in the incidence of labored breathing in all treated groups. No other meaningful differences in maternal antemortem and necropsy observations, body weight gain, food consumption or Cesarean section observations relative to those of the control group were seen at 1,200 mg/kg/day or less. No evidence of an adverse effect on fetal morphology was seen at any tested level. In conclusion, the 1,200 mg/kg/day level was determined to be the "no observable effect level" with respect to developmental toxicity, including teratogenesis, in this study.