Fatty acids, C16-18, Bu esters

Toxicity to reproduction

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented published GLP guideline study.

Data source

Reference

Reference Type:

publication

Title:

The safety of ethyl oleate is supported by a 91-day feeding study in rats

Author:

Bookstaff, R.C. et al.

Year:

2004

Bibliographic source:

Regulatory Toxicology and Pharmacology 39: 202–213

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:

not specified

Limit test:

no

Test material

Test material information

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
- Source: Victorian Chemical, Victoria, Richmond, Australia
- Analytical purity: 80.6%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratory
- Strain as given in publication: Sprague-Dawley rats [Crl:CD(SD)IGS BR]
- 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: AIN-93G 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:
The test item was formulated into the diet. For this purpose, 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 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:

91 days

Frequency of treatment:

daily ad libitum feeding

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
2.0, 3.9, 6.1 g/kg bw/day
Basis:
other: females, mean dose value as calculated from the actual body weight and food consumption data and dietary target concentration of 3.3, 6.7 and 10 % in feed

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1.8, 3.6, 5.5 g/kg bw/day
Basis:
other: males, mean dose value as calculated from the actual body weight and food consumption data and dietary target concentration of 3.3, 6.7 and 10 % 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: Bookstaff, 2004, rat, 90d oral, 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):

GROSS PATHOLOGY: all animals were subjected to gross pathological examination

ORGAN WEIGHTS: the following organs were weighed (paired organs weighed together). Organ-to-body weight percentages and organ-to-brain weight ratios were calculated: adrenal (2), pituitary gland, brain, prostate, epididymides (2), spleen, heart, testis (2), kidney (2), thymus, liver, thyroid with parathyroid, ovary (2), uterus.

HISTOPATHOLOGY: histopathology was done in control and high dose group animals.
The following tissues (when present) from each animal, with the exception of testes, were preserved in 10% neutral-buffered formalin and slides prepared for histopathological examination. Testes were preserved in Bouins fixative.
Adrenal (2)
Aorta
Brain (cerebrum, cerebellum, and medulla)
Cecum
Cervix
Colon [proximal and distal (2)]
Duodenum
Epididymis (2)
Esophagus
Eye (2)
Femur with bone marrow (articular surface of
the distal end)
Harderian gland
Heart
Ileum (including Peyers patch)
Jejunum
Kidney (2)
Lacrimal gland (exorbital)
Liver
Lung with mainstem bronchi
Lymph node (mandibular and mesenteric)
Mammary gland (females)
Nasal turbinates
Ovary (2)
Pancreas
Pituitary gland
Prostate
Rectum
Salivary gland [mandibular (2)]
Sciatic nerve
Seminal vesicle (2)
Skeletal muscle (thigh)
Skin
Spinal cord (cervical, thracic, lumbar)
Spleen
Sternum with bone marrow
Stomach (nonglandular and glandular)
Testis [preserved in Bouins fixative for
sacrificed animals (2)]
Thymus
Thyroid with parathyroid
Tissues with macroscopic changes or alterations
(i.e., gross lesions)
Tongue
Trachea
Urinary bladder
Uterus with uterine horns
Vagina
Zymbals gland

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)

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.

HISTOPATHOLOGY: NON-NEOPLASTIC
Referring to reproduction organs, no histopathological changes of any relevance were reported.

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

5 500 mg/kg bw/day (actual dose received)

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