Reaction mass of 1,3-diacetoxypropan-2-yl 12-acetoxyoctadecanoate and 2,3-diacetoxypropyl 12-acetoxyoctadecanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-06-30 to 2004-03-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study performed in accordance with GLP regulations. Dose analysis conducted by Covance was in accordance with GLP, comparative non-GLP analysis was conducted by sponsor. A deviation from GLP requirements was noted in relation to storage of samples used for stability analysis of TS-ED 532 in diet formulations. This deviation was evaluated not to have any impact on the analyses performed in the study.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

736150-63-3

EC Number:

616-005-1

Cas Number:

736150-63-3

IUPAC Name:

736150-63-3

Details on test material:

- Name of test material (as cited in study report): TS-ED 532
- Lot/batch No.: 10102
- Expiration date of the lot/batch: No information

Test animals

Species:

rat

Strain:

other: Rat/Hsd:Sprague Dawley SD rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rat/Hsd:Sprague Dawley SD rats
- Age at study initiation: 4 to 6 weeks of age,
- Weight at study initiation: 124 to 165 g for the males and 92 to 121 g for the females.
- Fasting period before study: no information
- Housing: Male and female rats were individually housed in suspended, stainless-steel cages
- Diet (e.g. ad libitum): basal diet ad libitum; certified rodent diet (#8728CM, Harlan Teklad)
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: no information

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25oC
- Humidity (%): 30 to 70%
- Air changes (per hr): a minimum of 10 room air changes/hour,
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): A two-week batch of TS-EE 532 admixed with the diet was prepared approximately once every two weeks. Dose was administered ad libitum weekly for at least 90 days.
- Mixing appropriate amounts with (Type of food): Basal Diet; certified rodent diet (#8728CM, Harlan Teklad)
- Storage temperature of food: Room temperature protected from light

VEHICLE
- Justification for use and choice of vehicle (if other than water): NA
- Concentration in vehicle: NA
- Amount of vehicle (if gavage): NA
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity:
Two separate mixing procedures were utilized for dose preparations that were utilized for homogeneity sample collection. A set of three samples (approximately 20 g each) from batches prepared from each mixing procedure were taken from the top, middle, and bottom of the low- and high-dose diet preparations mixed for prestudy analysis and for administration during Weeks 1 and 2. Two samples (approximately 20 g each) were also collected from basal diet used during the above collection times. Two samples from each of the three samples of the low- and high-dose diet preparations and one of the two samples of the basal diet were shipped to the sponsor for analysis under non-GLP conditions. One sample from each set of three samples of the low- and high-dose diet preparations and one of the two samples of the basal diet was held and analyzed by Covance.

Stability:
Five sets of duplicate samples (approximately 20 g each) were collected from the low- and high-dose diet preparations mixed for prestudy analysis. The samples were stored in a freezer following preparation. The samples were not requested by the sponsor for analysis. Stability studies that included the concentrations used in this study were conducted by Covance..

Concentration Verification:
Three samples (approximately 50 g) were collected from all dose preparations for Weeks 1, 2, 5, 7, 9, 11, and 13. Two samples (approximately 20 g each) were also collected from the basal diet used for dose preparation. Two sets of samples of test article in diet and one set of samples of the basal diet were sent to the sponsor for analysis under non-GLP conditions. One set of samples of TS-ED 532 in diet and one set of samples of the basal diet were retained and analyzed by Covance.

Duration of treatment / exposure:

90-93 days

Frequency of treatment:

Continously through the diet (7 days/week)

Doses / concentrations

Remarks:

Doses / Concentrations:
0. 0.4, 1.2 and 3.6% corresponding to targeted dose levels of 0, 500, 1600, or 5000 mg/kg bw/day.
Basis:
nominal in diet

No. of animals per sex per dose:

Male and female Hsd:Sprague Dawley SD rats (80/sex) were obtained from Harlan Sprague Dawley, Madison, Wisconsin, on 30 June 2003. Animals were randomly assigned to the following groups based on weight.

Group No. of animals Dose concentration (%) Approx. Dose level (mg/kg bw/day)
1 (control) 20 (male) 20 (female) 0 0
2 (Low) 20 (male) 20 (female) 0.4 500
3 (Mid) 20 (male) 20 (female) 1.2 1600
4 (High) 20 (male) 20 (female) 3.6 5000

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dose selection was based on existing toxicological and physical/chemical data.
- Rationale for animal assignment (if not random): NA
- Rationale for selecting satellite groups: NA
- Post-exposure recovery period in satellite groups: No
- Section schedule rationale (if not random): NA

Positive control:

No positive control group included.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily (a.m. and p.m.) for mortality and signs of pain and distress. Additional findings were recorded as they were observed.
.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were done once prior to initiation of treatment and weekly during the study, and on the day of scheduled sacrifice. Abnormal findings (ranked/graded, if appropriate) or an indication of normal was recorded. The weekly detailed observations were made outside of the home cage and included, but were not limited to, changes in the skin, fur, eyes, mucous membranes; occurrences of secretions and excretions; and autonomic activity. Changes in posture and reactivity to handling were also recorded if observed. Changes in gait were assessed weekly by allowing the animal to walk freely to allow evaluation of gait.
Expanded clinical observations were conducted once during the end of the exposure period (during Week 12 or 13); all observations were assessed and recorded for twenty animals/sex/group. Each animal was evaluated during handling (Hand-Held Observations), in an open field (Open Field Observations), and assessed for sensory reactivity to stimuli (Elicited Behavior).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were taken prior to initiation of treatment, on the first day of treatment, and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes,
Food consumption was recorded weekly.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:Ophthalmic examinations were done prior to initiation of treatment (all animals) and during Week 13 for Groups 1 and 4. Animals were examined by a veterinarian using an indirect ophthalmoscope. The eyes were dilated with a mydriatic agent prior to examination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Clinical pathology samples were taken for haematology, coagulation, and clinical chemistry, on Days 30 and 60 and at terminal sacrifice.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Clinical pathology samples were taken for haematology, coagulation, and clinical chemistry, on Days 30 and 60 and at terminal sacrifice.

NEUROBEHAVIOURAL EXAMINATION: Yes, Expanded clinical observation were conducted once during the end of the exposure period (during Week 12 or 13); all observations were assessed and recorded for twenty animals/sex/group. Each animal was evaluated during handling (Hand-Held Observations), in an open field (Open Field Observations), and assessed for sensory reactivity to stimuli (Elicited Behavior). Motor activity testing was also done once towards the end of the exposure period (during Week 12 or 13). Twenty animals/sex/group were placed into an automated photocell activity recording device for 40 minutes; activity counts were recorded at 2-minute intervals.


OTHER
- Faecal sample was collected during Weeks 11. Faeces from each selected animal were collected onto dry ice daily for 5 days. Individual faecal samples for each animal were weighed and the weight recorded daily of the five consecutive days. The faecal samples were shipped to the sponsor for analysis.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, After at least 90 days of treatment, all surviving animals were fasted overnight, bled for clinical pathology, then anaesthetized with sodium pentobarbital, exsanguinated, and necropsied. Organ weights were recorded at the scheduled sacrifice. Specified tissues were preserved in 10% neutral-buffered formalin unless otherwise noted. Tissue collected from the low- and mid-dose groups were held for possible future analysis (based on any findings in the high-dose group).

HISTOPATHOLOGY: Yes, All tissues collected from animals in the control and high-dose groups were processed and examined microscopically. Because there were no TS-ED 532 related findings in the high-dose group, the low- and mid-dose group tissues were not examined.

Other examinations:

Following collection of liver sections for histopathology, the remaining liver tissue from each animal was blotted to remove excess moisture, placed into appropriately labeled plastic sample bags, flash-frozen in liquid nitrogen, and stored in a freezer. The samples were used for peroxisome proliferation analysis.

Statistics:

The following statistical methods were used to analyze motor activity, grip strength, nociceptive reflex, body weight, body weight change, food consumption, continuous clinical pathology, and organ weight data:

- Levene’s test for variance homogeneity (Levene, 1960; Draper and Hunter, 1969)

- Rank transformation of data when the variances were heterogeneous (p < 0.05)

- One-way analysis of variance [ANOVA (Winer, 1971)] on raw homogeneous or rank-transformed homogeneous/heterogeneous data to be evaluated at
p < 0.05

- Dunnett’s t-test for control versus treatment group mean comparisons if ANOVA showed significance (Dunnett, 1955 and 1964) For each sex, Groups 2 through 4 were compared with Group 1 (control) at the 5%, two-tailed probability level. Only data collected on or after the first day of treatment were analyzed
statistically.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
All but one animal survived to the scheduled sacrifice and the one death of a female given 1600 mg/kg bw/day at an unscheduled interval was not considered to be TS-ED 532 related. No remarkable clinical observations were observed and there were no significant differences in body weight, body weight gain, or food consumption between the control and treatment groups with the exception of females given 1600 mg/kg bw/day (Group 3). Group 3 females were shown to have significant increases in body weight during Weeks 6 and 7 and food consumption during Weeks 5, 6 and 7. However, these findings were not considered adverse or TS-ED 532 related. Ophthalmic observations after 13 weeks of treatment did not reveal any visible eye lesions.

BODY WEIGHT, FOOD CONSUMPTION AND WEIGHT GAIN:
No remarkable clinical observations were observed and there were no significant differences in body weight, body weight gain, or food consumption between the control and treatment groups with the exception of females given 1600 mg/kg/day (Group 3). Group 3 females were shown to have significant increases in body weight during Weeks 6 and 7 and food consumption during Weeks 5, 6, and 7. However, these findings were not considered adverse nor TS-ED 532-related. During Week 1, consumption of TS-ED 532 by treatment groups ranged from 90 to 108% of the targeted levels of 500, 1600, and 5000 mg/kg/day for both males and females in Groups 2, 3, and 4, respectively. As expected, the mg/kg/day levels dropped throughout the study because diet concentrations remained fixed at 0.4, 1.2, and 3.6%; body weight increased; and food consumption did not increase nearly to the extent that body weight increased. The mean test article consumption over the 13 week period for Groups 2, 3, and 4 was 296, 881, and 2624 mg/kg/day, respectively, for males and 325, 1000, and 2964 mg/kg/day, respectively, for females.

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmic observations after 13 weeks of treatment did not reveal any visible eye lesions.

CLINICAL CHEMISTRY AND HAEMATOLOGY:
Although there were several statistically significant or otherwise notable differences for clinical pathology test results, most of the differences were considered incidental because they were extremely small and inconsistent over time (i.e., only observed at one testing interval) and between sexes or because they exhibited no relationship to dose. The only differences considered possibly test article-related were minimally higher alkaline
phosphatase for males and females given 5000 mg/kg/day. At Days 30 and 60, these minor differences were statistically significant for the females. By Day 93, the differences were less apparent. Regardless of their relationship to the test article, the findings for alkaline phosphatase were not considered toxicologically meaningful.

NEUROBEHAVIOUR:
No obvious TS-ED 532 related effects were revealed from the battery of neurological clinical observations that were made. No significant differences between control and treatment groups were observed for either forelimb or hindlimb grip strength or timed response to stimuli. Compared to control males, motor activity was significantly increased for Group 2 and Group 4 males during the 30-40 minute interval and for Group 3 and Group 4 males over the entire 0 to 40 minute period of testing. However, the increase in motor activity is not likely to be biologically relevant for the following reasons:

1. The increase was relatively small (21%) for Groups 3 and 4 compared to controls for mean motor activity over the entire 40 minutes;

2. The increase was not consistent. The difference between male control and treatment groups was not significant for all but the 30 to 40 minute interval, during which the controls became less active whereas treated males maintained the same activity. Also, females were not different at any interval, including the entire 0 to 40 minute period of testing;

3. There were no other abnormal behaviors observed in treated animals to correlate with an increase in motor activity. This includes assessment during standard clinical observations conducted throughout the treatment period as well as detailed neurobehavioural examinations performed near the end of treatment (i.e. expanded clinical observations which were performed during Week 13 along with the motor activity testing);

4. The increase in motor activity for treated males did not exhibit a clear dose-response relationship. Mean motor activity for Groups 3 and 4 were both increased 21% compared to control males despite the fact that Group 4 animals were dosed at 3-fold higher levels than Group 3 animals. And again, motor activity was not significantly different between female control and treatment groups.

ORGAN WEIGHTS:
Comparison of control versus 5000 mg/kg/day males and females showed there were no meaningful statistically significant organ weight differences, nor were there test articlerelated macroscopic or microscopic observations. Because of the absence of findings in the high-dose group, tissues from the two lower dose groups were not examined.

GROSS PATHOLOGY:
Although there were several statistically significant or otherwise notable differences for clinical pathology test results, most of the differences were considered incidental because they were extremely small and inconsistent over time (i.e., only observed at one testing interval) and between sexes or because they exhibited no relationship to dose.

HISTOPATHOLOGY: Comparison of control versus 5000 mg/kg/day males and females showed there were no meaningful statistically significant organ weight differences, nor were thereTS-ED 532 related macroscopic or microscopic observations. Because of the absence of findings in the high-dose group, tissues from the two lower dose groups were not examined.

HISTORICAL CONTROL DATA (if applicable). NA

OTHER: Metabolism of TS-ED 532 was characterized by analyzing the non-absorbed 12-hydroxystearic acid components in the feces. No unhydrolyzed TS-ED 532 was detected. Results of fecal analysis from males and females fed 3.6% test article indicated there was not a sex-specific difference in metabolism. Comparison of free versus acetylated 12-hydroxystearic acid indicated that the free acid form increased more than the acetylated free acid form as dose increased. When excretion was expressed relative to amount consumed, the excretion rate appeared to decrease as TS-ED 532 consumption increased.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 5 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Results of stability analysis performed by the sponsor (Danisco) at the high and low concentrations used on this study showed that TS-ED 532 was stable under the conditions of use on this study. Dose analysis (homogeneity and concentration verification) was initially performed by sponsor. Because sponsor’s laboratory was not compliant with GLP requirements, a decision was made to analyze the back-up dose analysis samples under GLP compliant conditions at Covance.

The back-up dose analysis samples were stored at Covance for approximately 4 months at -60 to -80 °C prior to analysis. Because neither sponsor nor Covance has stability information to support such storage, this is a deviation from GLP requirements. However, TS-ED 532 diet formulations were shown to be relatively stable at 4 °C, and expected to be much greater at -60 to -80 °C. Furthermore, dose analysis results for all but the low concentration were sufficiently close to target, which indicates that the initial TS-ED 532 content would not have been significantly below the target concentration. Finally, the sponsor performed non-GLP analysis of the dose analysis samples soon after the diets were prepared and the results were all acceptably close to target.

Applicant's summary and conclusion

Conclusions:

A 90-days toxicity study was performed using TS-ED 532 admixed to the diet at concentrations of 0.4, 1.2, or 3.6% (corresponding to approx. dose levels of 500, 1600 and 5000 mg/kg bw/day, respectively). Orally administered TS-ED 532 for at least 90 days did not result in adverse signs of toxicity hence the NOAEL was established as > 5000 mg/kg bw/day in both sexes, based on no adverse treatment-related effects at the highest dose tested.

Executive summary:

A 90-days study (OECD 408) was performed using TS-ED 532 admixed to the diet at concentrations of 0.4, 1.2, or 3.6% (corresponding to approx. dose levels of 500, 1600 and 5000 mg/kg bw/day, respectively).

No significant differences were observed in body weight, body weight gain, or food consumption between the control and treatment groups with the exception of females given 1600 mg/kg bw/day. These females were shown to have significant increases in body weight during weeks 6 and 7 and food consumption during weeks 5, 6 and 7. However, these findings were not considered adverse or TS-ED 532-related. Higher alkaline phosphatase for males and females given 5000 mg/kg bw/day were observed at days 30 and 60 but were only statistically significant for the females and were not present at the next observation period.

The functional observations revealed no treatment-related effects in any dose group for either sex. A slight dose-dependent trend towards decreased mean response time in males was observed but was not found to be statistically significant. Based on the lack of statistical significance and the absence of this difference in females, the observation was not considered to be treatment-related. Haematology and clinical chemistry observations showed no treatment-related patterns. A statistically significant increase in alkaline phosphatase was observed at days 30 and 60 for females given 5000 mg/kg bw/day, however, the increase was well within the historical range in rats and was not considered toxicologically meaningful. There were no ophthalmological findings observed in either sex at any dose. No treatment-related macroscopic observations were noted. Absolute and relative liver weights were increased in the females dose with 1600 mg/kg bw/day. Histopathological findings were not significant and did not correlate with organ weight findings. In the absence of related findings, the changes in organ weights were considered adaptive in nature.

Orally administered TS-ED 532 for at least 90 days did not result in adverse signs of toxicity. Hence a NOAEL was established as > 5000 mg/kg bw/day in both sexes, based on no adverse treatment-related effects at the highest dose tested.