Registration Dossier

Diss Factsheets

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: extended 90 day feeding study with fertility parameters
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented published GLP Guideline study.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: 1993 FDA draft "Redbook II" guidelines (Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food).
Principles of method if other than guideline:
The purpose of this study was to determine the safety of ethyl oleate (EO) in a 91-day feeding study in Sprague-Dawley rats.
Additionally to the repeated dose toxicity, oestrus cycle and sperm parameters were analyzed.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Ethyl oleate
EC Number:
203-889-5
EC Name:
Ethyl oleate
Cas Number:
111-62-6
IUPAC Name:
ethyl octadec-9-enoate
Details on test material:
- Name of test material (as cited in study report): Ethyl oleate (EO); 9-octadecenoic acid ethyl ester
- CAS No. of test material (as cited in study report): 111-62-6
- Source: Victorian Chemical, Victoria, Richmond, Australia
- Analytical purity: 80.6 %
Analytical testing showed it complied with both National Formulary (NF) and European Pharmacopoeia (EP) specifications.
The EO used in this study is oil derived from the ethylation of sunflower oil. To be compliant with the NF and EP monographs for EO, the fraction of the oil that is actually the EO molecule must be at least 60%. The EO used in this study was analyzed by GC to determine fatty acid composition, and by HPLC to determine exact concentration of EO (determined to be 80.6%). Table 1 shows the fatty acid composition (as percent of total fatty acids) of both the EO oil and the HOSO, for those fatty acids 0.1% or greater. For EO, the fatty acids are in the form of ethyl esters and for HOSO, the fatty acids are as triglycerides.

Test animals

Species:
rat
Strain:
other: Sprague–Dawley rats [Crl:CD(SD)IGS BR]
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory
- Age at study initiation: approx. 5-6 weeks
- Weight at study initiation: approx. 150–175 g
- Fasting period before study: one night prior to blood collections
- Housing: individually in stainless steel cages
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
Temperature and humidity were controlled throughout the duration of the study.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: diet containing high oleic safflower oil (HOSO)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test materials were formulated into a purified diet based on the AIN-93G purified diet. The AIN-93G diet was modified to allow incorporation of an additional 10% of test fat (either EO, HOSO, or a combination of the two). The modification involved decreasing overall carbohydrate concentration to allow the incorporation of an additional 10% of test fat without diluting out other nutrients. The composition of this basal diet is shown in Table 2.

In this study, the test diets were prepared based on the addition of the EO oil (i.e., the high-dose diets contained 10% of the EO oil), and were not adjusted based on the actual concentration of the EO molecule.

VEHICLE
- Source: High oleic safflower oil (HOSO) was obtained from Columbus Foods Company, Chicago, Illinois.
Details on mating procedure:
no matings performed
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Data on test material stability, homogeneity, and diet concentrations showed that the test material was stable over the course of the study, and diet concentrations were homogeneous and within 10% of target (data not shown).
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily ad libitum feeding
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2.0, 3.9, 6.1 g/kg/day
Basis:
other: females, calculated from actual body weight and food consumption data and target concentration of ethyl oleate in feed
Remarks:
Doses / Concentrations:
0, 1.8, 3.6, 5.5 g/kg/day
Basis:
other: males, calculated from actual body weight and food consumption data and target concentration of ethyl oleate in feed
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
see 7.5.1: key, Bookstaff, 2004, 90d oral rat, RL2
Oestrous cyclicity (parental animals):
Beginning on the first day of Week 11 and continuing for 21 consecutive days, all females had daily vaginal smears prepared and examined to evaluate the stage of the estrous cycle.
Sperm parameters (parental animals):
Parameters examined: testis weight, epididymis weight, sperm count in epididymides, sperm motility, sperm morphology.
At the terminal sacrifice, males were evaluated for sperm viability.
- For motility and morphology assessment, the right vas deferens was excised and immediately placed in a petri dish containing 10 ml of a 1% bovine serum albumin dissolved in phosphate buffered saline. The solution was prewarmed to approximately 38 C. A 3- to 4-min period (minimum to maximum period, respectively) was allowed for the sperm to swim out. Following the swim-out period, a sample of sperm was collected using a 100-micron-deep cannula and immediately loaded into a prewarmed stage of the Hamilton Thorne IVOS automated sperm analyzer. Five fields/animal were selected and stored as digital images. These images were analyzed for percent motility.
- Sperm morphology was assessed with two slides of sperm stained with eosin for each male. A minimum of 200 sperm cells was evaluated.
- For sperm count, the right epididymis was removed and divided in half by cross sectioning through the middle. The tail end caudal section was placed on dry ice, and stored frozen at approximately -60 to -80 C until analysis for total sperm count.
Postmortem examinations (parental animals):
see 7.5.1: key, Bookstaff, 2004, 90d oral rat, RL2
Statistics:
Control versus treated group comparisons (Groups 2 through 4 versus Group 1) were evaluated at the 5.0%, two-tailed probability level. Data for each sex were analyzed separately. If Levene_s test for variance homogeneity was not significant (p > 0.05), one-way analysis of variance (ANOVA) was performed on the observed values. If Levene's test was
significant (p < 0.05), ANOVA was done on the rank transformed data. Post-hoc Dunnett's t-test was used for control versus treated group mean comparison, incorporating transformations when necessary.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: decreased food intake due to lower palatability

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
see 7.5.1

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
see 7.5.1

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
see 7.5.1

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no treatment-related effects on reproductive capacity as evaluated by female estrous cycle.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no treatment-related effects on reproductive capacity as evaluated by sperm motility, sperm count, or sperm morphology.

ORGAN WEIGHTS
The only statistically significant effects were a decrease in terminal body weight in the Group 3 and 4 females vs. Group 1, and a slight increase in brainto-body weight percent in the Group 3 and 4 females, which is driven by the decrease in terminal body weight seen in these groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
Hepatocellular vacuolation typical of fat accumulation was noted for both control and high-dose animals. The incidence and severity of the vacuolation were higher for animals given 10% HOSO (controls) than for the animals given 10% EO. Evaluation of other organs /tissues did not reveal any test
article-related findings.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
ca. 6 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: oestrus cyclus in females, sperm characterization in males and histologic examinations (incl. Epididymides, Mammary gland, Ovaries, Prostate, Seminal vesicles, Testes, Thyroid with parathyroid, Uterus with uterine horns and Vagina)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion