C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-03-04 to 2003-04-02

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

EC Number:

701-479-5

Molecular formula:

C42H82O7

IUPAC Name:

C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

Test material form:

other: clear to slightly hazy, viscous liquid

Details on test material:

- Name of test material (as cited in study report): DERMOL DGDIS
- Physical state: clear to slightly hazy, viscous liquid
- Composition: Diglyceryl Diisooctanoate
- Analytical purity: n.a.
- Lot/batch No.: #157-67
- Expiration date of the lot/batch: n.a.
- Stability under test conditions: Expected to be stable for the duration of testing

Test animals

Species:

rat

Strain:

other: CRLCD (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raliegh, N.C.
- Age at study initiation: about 9 weeks
- Weight at study initiation: range from 280- 350 gram (males); 207-244 gram (females)
- Housing: rats were single housed
- Diet: ad libitum
- Water : ad libitum
- Acclimation period: Approx. 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26 °C
- Humidity (%): 35-70 %
- Air changes (per hr):
- Photoperiod: 12-hr light/dark cycle

IN-LIFE DATES: From: 2003-03-04 To: 2003-04-02

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): tree times during study (Pre-test, test days 3 and 13)
- Mixing appropriate amounts with (Type of food): standard Rat Chow
- Storage temperature of food: refrigerated at approximately 5°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): none

The test and control diets were presented to each group of rats on day 0 of study. Enough diet was provided each week to insure ad-libitum feeding, except during the 18-hours fasting period prior to blood collection. All animals were exposed to the test or control diets for 28 days (day 0 to day 28)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formula: Average daily food consumption, mg)(Concentration of Test Substance in Diet) devided by Average Body Weight, kg.
Samples (about 75 g) of each concentration of test diet were obtained from middle of the mixing bowl for all preparations. A control diet sample was obtained from the third preparation. All samples were labeled with study number, test groups nominal concentration and date of preparation and stored frozen for analysis. Not presented in report.

Duration of treatment / exposure:

All animals were exposed to the test or control diets for 28 days (day 0 to day 28)

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.012 %: Targeted dose level = 10 mg/kg/day Low dose; Average Dose Level: male 9 mg/kg/day, female 10 mg/kg/day
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.12 %: Targeted dose level = 100 mg/kg/day intermediate dose; Average Dose Level: male 91 mg/kg/day, female 100 mg/kg/day
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1.2 %: Targeted dose level = 1,000 mg/kg/day high dose; Average Dose Level: male 845 mg/kg/day, female 922 mg/kg/day
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
0 %: Targeted dose level = mg/kg/day Control; Average Dose Level: male 0 mg/kg/day, female 0 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

5 /group/sex , (total 20 males and 20 females)

Control animals:

yes, plain diet

Details on study design:

Dietary concentratios of 0. 0.012 , 0.12 and 1.3 % were administered to the animals to ahive the targeted dose levels. The human exposure to DERMOL DGDIS is expected to be approximately 0.7 mg/kg/day. The levels selected for this study are at least 10 x, 100 x and 1000 x the expected human exposure.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Cage-side observations were made daily during the study. Observations included, but were not limitcd to, gross evaluation of skin, fur, eyes, mucous membranes, occurrences of secretions and excretions, respiration, circulation, autonomic and central nervous systems, somatomotor activity and behavior patterns. Particular attention was directed to changes in gait and posture, and increased salivation.
- Time schedule: twice daily for mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly (days 0, 7, 14, 21, and 28) on all animals assigned to study.
For these evaluations, each animal was removed from its cage so that its reaction to handling and its behavior in an open field would be assessed.The observation recorded for each animal included, but was not limited to, changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Particular attention was directed to changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, sterotypies (e.g., excessive
grooming, repetitive circling), difficult or prolonged parturition or bizaITe behavior (e.g., selfmutilation, walking backwards).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded twice during the acclimation period, prior to study initiation
(Day 0) and then weekly (Days 7, 14, etc.) thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, individual food consumption was measured and recorded weekly.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The average daily dietary intake of the test item was also calculatcd.

FOOD EFFICIENCY:
- Food efficiency was calculated by dividing the body weight gain by the food consumption over equivalent periods.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Yes, under isoflurane anesthesia
- Animals fasted: Yes, 18 hours prior to blood collection
- How many animals: all
- Parameters checked: hemoglobin concentration (HGB), mean corpuscular volume (MCV), erytlu'oeyte count (REC), total white blood cell (WaC) and differential leukocyte count, platelet count (PLT), red cell distribution width (RDW) prothrombin time (PT) and activated partial thromboplastin time (APTT). Absolute reticulocyte count (ARET), hematocrit (HCT), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were determined. Blood smears were prepared and evaluated.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Animals fasted: Yes, 18 hours prior to blood collection
- How many animals: all
- Parameters checked: calcium (CALC), inorganic phosphorus (IPHS), chloride (CL), sodium (NA), potassium (K), fasting glucosc (GLUC), serum alanine aminotransferase (ALT), serum aspartatc aminotransferase (AST), sorbital dehydrogenase (SOH), alkaline phosphatase (ALKP), urea nitrogen (BUN), albumin (ALB), blood creatinine (CREA), total bilirubin (BILl), total serum protein (TP), globulin (GLOB), total cholesterol (CHOL), and triglycerides (TRlG).

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On Day 29, all animals were euthanized by exsanguination from the abdominal aorta under isoflurane anesthesia. All animals were subjected to a full necropsy, which included examination of the extenal surface of the body, all orifices and the thoracic, abdominal and cranial cavities and their contents. The liver, kidneys, adrenals, brain, heart, thymus, spleen, testes and epididymides (of all animals sacrificed by design) were weighed wet as soon as possible after dissection to avoid drying.
The following organs and tissues from all animals were preserved in 10% neutral buffered formalin for possible future histopathological examination: lungs, brain-including sections of medulla/pons, cerebellar cortex and cerebral coltex, spinal cord (3 levels: cervical, midthoracic, and lumbar), pituitary, thyroid, thymus, trachea, heart, sternum with bone marrow, liver, spleen, kidneys, adrenals, pancreas, ovaries, testes, uterus, vagina, accessory genital organs (epididymides, prostate, and seminal vesicles), stomach, duodenum,jejunum, ileum, cecum, colon, rectum, urinary bladder, reprcsentativc lymph node, and peripheral nerve (sciatic).

HISTOPATHOLOGY: Yes
Histological examination was performed on the preserved organs and tissues of the animals from the control (Groups I and 2) and high dose (Groups 7 and 8) groups. The fixed tissues were trimmed, processed, embedded in paraffin, microtomed, placcd on glass microscope slides, and stained with hematoxylin and eosin. Slide preparation and histopathological assessments were performed by Experimental Pathology Laboratories, Inc. (EPL®).

Statistics:

Mean and standard deviations were calculated for body weight, body weight gain, food consumption, food efficiency, organ weight and organ to body/brain weight ratio data.
Treated and control groups were compared using a One-Way of Analysis (ANOVA), followed by comparison of the treated groups to control by Dunnett's Multiple Comparisons test.
Data was evaluated for homogeneity of variances and normality by the Bartlett's test, and Kolmogrov and Smimov tests, respectively. Data that was considered by Bartlett's test was further run with a non-parametric method (Dunn's test). (INSTAT Biostatistics, Graph Pad Software, San Diego, CA). Significanee was judged at p <0.05. Male and female rats were evaluated separately.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities during this study. All animals appcarcd active and healthy. There were no signs of gross toxicity, adverse pharmacologic effects or abnormal behavior.

BODY WEIGHT AND WEIGHT GAIN
Although one statistical difference in weekly body weight gain was noted between Group 8 females and Group 2 controls during weekk 1, there were no other statistically significant differences (p >0.05) in body weight or body weight gains between any test and their corresponding control groups. Thus, body weight was considercd not affected by thc dietary administration of the test item.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Although statistical differences in weekly food consumption and food efficiency were noted between Group 8 females and Group 2 controls during Week 1, there were no other statistically significant differences (p > 0.05) in food consumption or food efficiency between any test and their corresponding control groups. Thus, no adverse effects in these parameters were produced by the dietary administration of the test item. The average daily intake of the test item in male rats fed dietary concentrations of 0%, and approximately 0.012%, 0.12% and 1.2% was 0, 9, 91 and 845 mg/kg/day, respectively. For the same dietary concentrations, the average daily intake of the test item in female rats was 0, 10, 100 and 922 mg/kg/day, respectively.

HAEMATOLOGY and CLINICAL CHEMISTRY
There were no adverse or treatment-related changes in hematology, coagulation, or clinical chemistry parameters in male or female rats.

ORGAN WEIGHTS
There were no adverse or treatment-related changes with the exception of the increased kidney- and liver-weights (male and female) and increased liver-weight in the high dose female group. The liver weight increases are likely indicative of a normal physiological compensatory response to a high level oral exposure (i.e., significantly execeds the expected human exposure) of a xenobiotic. There were no histological alteration or changes in clinical pathology inticators observed.

GROSS PATHOLOGY
There were no treatment-related macroscopic changes present in this study.

HISTOPATHOLOGY: NON-NEOPLASTIC
There was no histologic correlation to the increases in liver weight noted in treated rats. All other microscopic lesions were of the type and incidence commonly noted in Sprague-Dawley rats.

HISTOPATHOLOGY: NEOPLASTIC (if applicable) not observerd.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

845 mg/kg bw/day (nominal)

Based on:

other: Since no analyses of diets were performed, these values are based on nominal concentrations of the test item in diets.

Sex:

male

Basis for effect level:

food efficiency

2

Key result

Dose descriptor:

NOAEL

Effect level:

922 mg/kg bw/day (nominal)

Based on:

other: Since no analyses of diets were performed, these values are based on nominal concentrations of the test item in diets.

Sex:

female

Basis for effect level:

food efficiency

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study and based on the toxicological endpoints evaluated, the No Observed-Adverse-Effect Level (NOAEL) the test item was considered to be 845 mg/kg/day for male rats and 922 mg/kg/day for female rats.

Executive summary:

A 28-day dietary study was performed in Sprague Dawley rats to determine the potential of the test item to produce systemic toxicity. Eight groups of five rats (male or female only) each were presented with a diet containing 0% (Groups 1 and 2), and approximately 0.012% (Groups 3 and 4), 0.12% (Groups 5 and 6) or 1.2% (Groups 7 and 8) of the test substance. Targeted dietary intake levels of the test substance were 0, 10, 100, and 1,000 mg/kg/day. Animals were observed daily for clinical signs and mortality. Individual food consumption and body weights were recorded weekly. Blood was sampled from all animals on Day 29 of the study for hematology and clinical chemistry assessments. Gross necropsies were performed on all animals at terminal sacrifice and organs and tissues were evaluated histologically.

There were no mortalities, clinical signs, body weight or nutritional effects, clinical pathology, or gross or histopathology alterations that were considered related to the dietary administration of the test substance and/or considered to be of toxicological significance. There were no significant effects in rats of either sex after 28 consecutive days of the test item presented in the diet. Based on the parameters evaluated, the No-Observed-Adverse-Effect Level (NOAEL) for this study is considered to be 845 mg/kg/day in males and 922 mg/kg/day in females, the highest dose tested.