Fatty acids, C16-18 and C18-unsatd., Et esters

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction

Remarks:

other: extended 90 day feeding study with fertility parameters

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented published GLP guideline study.

Reason / purpose for cross-reference:

reference to same study

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

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.

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

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

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

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.

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

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.

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)

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 2) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional group(s), common precursors/breakdown products and similarities in physicochemical and toxicological properties (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for grouping of substances and read-across

There are no data available for the reproduction toxicity of Fatty acids, C16-18 and C18-unsatd., ethyl esters (CAS# 85049-36-1). In order to fulfil the standard information requirements set out in Annex IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, a read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

The general rules for grouping of substances and read-across approach were followed, as laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

 

Overview Reproductive toxicity:

CAS#	Reproductive Toxicity
85049-36-1 Target substance	RA: 111-62-6
111-62-6	NOAEL: 5500 mg/kg bw/day

The above-mentioned substance is considered to be similar on the basis of structural similarity resulting in similar properties and/or activities. The available endpoint information is used to predict the outcome of the same endpoints for Fatty acids, C16-18 and C18-unsatd., ethyl esters (CAS 85049-36-1).

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

NOAEL (fertility) = 5500 mg/kg bw/day, OECD 408

A 90-day oral feeding study (Bookstaff, 2004) was performed with Ethyl Oleate (CAS# 111-62-6) according to the 1993 FDA draft "Redbook II" guidelines (Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food). The study was performed equivalently to OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents) with additional assessments of oestrus cycle and sperm parameters. The purpose of the study was to determine the safety of Ethyl Oleate (EO) in a 91-day feeding study in Sprague-Dawley rats. EO was mixed into AIN-93G purified diet at levels of approx. 0, 1900, 3800 and 6000 mg/kg bw/day. All the diets were calorie- and fat-matched using high oleic safflower oil (HOSO) as the control fat. There were 20 male and 20 female rats per group. EO in the diet was well tolerated and there were no toxicologically significant findings in any of the measured parameters (clinical observations, body weight gains, appearance of the faeces, ophthalmic examinations, haematology, clinical chemistry, urinalysis, organ weights, histopathology, or male and female reproductive assessments). Based on the absence of abnormalities concerning oestrus cyclus, sperm characterization and histopathologic evaluation of oestrus cycle 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) the subchronic 90-day oral NOAEL for fertility in rats for Ethyl Oleate was found to be approx. 5500 mg/kg bw/day.

Conclusion for reproduction toxicity

One study is available investigating the reproduction toxicity using analogue based read-across. The study from the analogue substance ethyl oleate (CAS 111-62-6) did not show treatment-related effects up to the highest tested dose level. Thus, no hazard for reproduction toxicity was identified.

Short description of key information:
No hazard for reproductive toxicity was identified for Fatty acids, C16-18 and C18-unsatd., ethyl esters.

Justification for selection of Effect on fertility via oral route:
Hazard assessment is conducted by means of read-across. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties and overall quality assessment (refer to the endpoint discussion for further details).

Effects on developmental toxicity

Description of key information

The NOAEL for maternal and developmental toxicity for 2-ethylhexylstearate (CAS 91031-48-0) and for 2-ethyl hexyl stearate (CAS 22047-49-0) was found to be 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

Justification for grouping of substances and read-across

There are no data available for the reproduction toxicity of Fatty acids, C16-18 and C18-unsatd., ethyl esters (CAS 85049-36-1). In order to fulfil the standard information requirements set out in Annex IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, a read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

The general rules for grouping of substances and read-across approach were followed, as laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

 

Overview of developmental toxicity

CAS#	Toxicity
22047-49-0 (a)	NOAEL: 1000 mg/kg bw/day
91031-48-0 (a)	NOAEL: 1000 mg/kg bw/day

 

The above-mentioned substance is considered to be similar on the basis of structural similarity resulting in similar properties and/or activities. The available endpoint information is used to predict the outcome of the same endpoints for Fatty acids, C16-18 and C18-unsatd., ethyl esters (CAS 85049-36-1).

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

NOAEL (fertility) = 5500 mg/kg bw/day, OECD 408

A 90-day oral feeding study (Bookstaff, 2004) was performed with Ethyl Oleate (CAS 111-62-6) according to the 1993 FDA draft "Redbook II" guidelines (Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food). The study was performed equivalently to OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents) with additional assessments of oestrus cycle and sperm parameters. The purpose of the study was to determine the safety of Ethyl Oleate (EO) in a 91-day feeding study in Sprague-Dawley rats. EO was mixed into AIN-93G purified diet at levels of approx. 0, 1900, 3800 and 6000 mg/kg bw/day. All the diets were calorie- and fat-matched using high oleic safflower oil (HOSO) as the control fat. There were 20 male and 20 female rats per group. EO in the diet was well tolerated and there were no toxicologically significant findings in any of the measured parameters (clinical observations, body weight gains, appearance of the faeces, ophthalmic examinations, haematology, clinical chemistry, urinalysis, organ weights, histopathology, or male and female reproductive assessments). Based on the absence of abnormalities concerning oestrus cyclus, sperm characterization and histopathologic evaluation of oestrus cycle 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) the subchronic 90-day oral NOAEL for fertility in rats for Ethyl Oleate was found to be approx. 5500 mg/kg bw/day.

Conclusion for reproduction toxicity

One study is available investigating the reproduction toxicity using analogue based read-across. The study from the analogue substance ethyl oleate (CAS 111-62-6) did not show treatment-related effects up to the highest tested dose level. Thus, no hazard for reproduction toxicity was identified.

Justification for classification or non-classification

Based on read-across from the structurally similar substances, the available data on toxicity to reproduction and development do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.