Fatty acids, C18 unsaturated, ethyl & methyl esters

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies, from source substances with similar structure and intrinsic properties. Read-across is justified based on common functional group, common precursors/breakdown products and similarities in PC/ECO/TOX properties. Please refer to the endpoint discussion for further details.
The selected studies are 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

Toxicity to reproduction

Justification for read-across

There are no data regarding reproduction toxicity available for Fatty acids, C18 unsaturated, ethyl & methyl esters. In order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex IX, 8.7, read-across to appropriate substances is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5.

According to 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) “to avoid the need to test every substance for every endpoint”.

Fatty acids, C18 unsaturated, ethyl & methyl esters is a multi-constituent substance specified by esters of ethanol and methanol with fatty acids C18 unsaturated (source canola oil) resulting in monoesters which meets the definition of an UVCB substance. Thus, the test substance represents fatty acid esters which undergo to a high extent hydrolysis by ubiquitous expressed gastrointestinal enzymes into the free fatty acid components and the respective alcohol moieties (Lehninger, 1970; Mattson and Volpenhein, 1972). Considering the common metabolism, the read-across approach is based on the presence of a common functional group, common precursors and the likelihood of common breakdown products via biological processes, which result in structurally similar chemicals with similar physico-chemical, toxicological and toxicokinetic behaviour. For further details on the read-across approach, please refer to the analogue justification in section 13 of the technical dossier.

As no data on reproduction toxicity are available for Fatty acids, C18 unsaturated, ethyl & methyl esters, read-across to reliable data on the analogue substances Ethyl oleate (CAS 111-62-6), Methyl laurate (CAS 111-82-0) and Butyl stearate (CAS 123-95-5) was conducted. Ethyl oleate (CAS 111-62-6) is the main component of Fatty acids, C18 unsaturated, ethyl & methyl esters. Methyl laurate (CAS 111-82-0) and Butyl stearate (CAS 123-95-5) are also fatty acid esters which are composed of methanol or butanol (alcohol moiety) and lauric acid (C12 fatty acid moiety) or stearic acid (C18 fatty acid moiety), respectively. Therefore, they are regarded as structurally similar to Fatty acids, C18 unsaturated, ethyl & methyl esters and are hence considered as suitable analogue substances.

CAS 111-62-6

A 91-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 in Rodents) with additional assessment of oestrus cycle and sperm parameters. The purpose of the study was to determine the safety of Ethyl oleate in a 91-day feeding study in Sprague-Dawley rats. Ethyl oleate was mixed into AIN-93G purified diet at levels of approx. 0, 1900, 3800 and 6000 mg/kg bw/day. All 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. Ethyl oleate 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, hematology, clinical chemistry, urinalysis, organ weights, histopathology, or male and female reproductive assessments). Based on the absence of abnormalities concerning oestrus cyclus, 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 91-day oral NOAEL for fertility in rats for Ethyl oleate was found to be approx. 5500 mg/kg bw/day.

CAS 111-82-0

The substance Methyl laurate (CAS 111-82-0) was studied for oral toxicity in rats in an OECD Guideline 422 combined repeat dose and reproductive/developmental toxicity screening test under GLP conditions (Tanaka, 2000). 12 male and female Crj:CD (SD) rats per dose were administered doses of 0, 250, 500 and 1000 mg/kg/day by gavage. The test material was administered to females from 14 days before mating until day 3 of lactation and to males for 45 days. Terminal kill was on day 45 for males and on day 4 of lactation for females. There were no effects of the test substance on the estrous cycle, copulation index, fertility index, gestation length, number of corpora lutea or implantations, implantation index, delivery index or parturition. The NO(A)ELs for reproductive performance of males and females, as well as pup development are considered to be ≥ 1000 mg/kg/day in each case.

CAS 123-95-5

A reproduction/developmental toxicity screening test was performed with Butyl stearate (CAS 123-95-5) (Smith, 1953). 20 male and female Sprague-Dawley rats received Butyl stearate at a concentration of 6.25% in the diet, corresponding to approx. 6000 mg/kg bw/day for a period of 10 weeks. Negative control animals (12 males and females) were fed with the concurrent basal diet. After 10 weeks animals were mated. Successfully mated pregnant females were housed individually in breeding cages. The date of parturition and the number and sex of pups in each litter were recorded. Litters were weaned 21 days postpartum and the weights of the weanlings determined. From each of the three groups of weanlings (those receiving the test material and the controls), 24 males and 24 females were chosen at random and for the next 21 days, these young were fed the same 6.25% diet as had been ingested by their parents. Diet intake and body weights were recorded daily; 21 days after weaning, the rats were sacrificed and necropsies were performed.

Based on reproduction, fertility index, litter size, survival index/viability index of offspring and necropsy at day 21 after weaning the NOAEL for parental fertility as well as for offspring development was found to be 6000 mg/kg bw/day.

Conclusion for reproduction toxicity

The available data show that the analogue substances do not possess intrinsic hazardous properties in regard to reproduction toxicity. Therefore, based on common functional groups and structural similarities, Fatty acids, C18 unsaturated, ethyl & methyl esters is considered to be non-hazardous regarding reproduction toxicity.

A waiver for the requirement to perform an extended one-generation reproduction toxicity study (standard configuration or with additional modules) is included in the dossier, as the study is not considered as scientifically necessary in regard to the available data on repeated dose toxicity and reproductive/developmental screening studies and, considering concerns regarding the use of vertebrate animals for experimental purposes, as unjustified.

A detailed reference list can be found in the CSR and in section 13 of the dossier.

Short description of key information:
Based on a weight of evidence approach, all available data resulted in NOAELs for fertility of ≥ 1000 mg/kg bw/day or higher.

Justification for selection of Effect on fertility via oral route:
No study was selected as hazard assessment is conducted by means of read-across from structural analogues. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances 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) was found to be 1000 mg/kg bw/day (OECD 414)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with acceptable restrictions (exposure duration was only from day 6-15 of gestation instead of day 5-19).

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2001

Deviations:

yes

Remarks:

Exposure duration was only from day 6-15 of gestation instead of day 5-19.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: approx. 8-10 weeks
- Weight at study initiation: mean approx. 197 g
- Housing: individually in Makrolon Type M3 cages (Ebeco) with standard softwood bedding (ARWI-Center, Essen, Germany)
- Diet: Pelleted Altromin Maintenance Diet 1324, Lot No. 221092/1558 (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 40-56
- Air changes (per hr): 10-15 per hr
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: Arachidis oil, DAB 10

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing solutions were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility of test substance
- Concentration in vehicle: 20, 60 and 200 mg/mL, respectively for the 100, 300 and 1000 mg/kg bw dose groups
- Amount of vehicle (if gavage): 5 mL/kg bw

After arrival all females were assigned to the different groups using a computer-generated random algorithm.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant, at day 0
- Proof of pregnancy: vaginal plug day 0 of pregnancy

Duration of treatment / exposure:

from day 6 up to day 15 of gestation

Frequency of treatment:

once daily

Duration of test:

until day 20 of gestation

No. of animals per sex per dose:

24 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Other:
group 1: 0 mg/kg bw/day
group 2: 100 mg/kg bw/day
group 3: 300 mg/kg bw/day
group 4: 1000 mg/kg bw/day

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
Mortality rate: The animals were checked at least twice daily for any mortality.
Signs and/or symptoms: The animals were observed at least twice daily (working days) for signs of reaction to treatment and/or symptoms of illness.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Post mortem examination, including gross macroscopic examination of all maternal organs, with emphasis on the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea, was performed and the data recorded.
The uteri (including content) of all females were weighed at necropsy on day 20 post coitum to enable the calculation of the corrected body weight gain.
- Any female sacrificed or found dead during the study was subjected to macroscopic examination of the visceral organs, with emphasis on the uterus and its content.

BODY WEIGHT: Yes, mean body weight changes
- Time schedule for examinations: days 0, 6, 16 and 20 of gestation

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Number and distribution of intrauterine implantations were classified as live or death fetuses, late intrauterine deaths (resorptions), early intrauterine (resorption sites). The fetuses were removed from the uterus. Intrauterine deaths were classified on the basis of the presence (late) or absence (early) of fetal or decidual tissue in addition to placental tissue.

Fetal examinations:

- External examinations: Yes: half per litter
- Soft tissue examinations: Yes: half per litter: malformations oh hydrocephalus, variations of brain, adrenal gland, renal pelvis, ureter
- Skeletal examinations: Yes: half per litter: malformations of hydrops, retardations of skull bones, hyoid, sternebrae, pelvis, 13th rib
- Head examinations: Yes: half per litter

The live fetuses were sexed, weighed individually including placentae, examined for gross external abnormalities and allocated to one of the following procedures:
1) Half of the fetuses from each litter was non individually fixed in Bouin's solution in order to examine viscera and brain by Wilson's slicing technique. After examination the sections were not preserved.
2) The remaining fetuses were placed non individually in a solution of potassium hydroxide for clearing and were stained with alizarin red (Shandon Varistain 24-T). The skeletons were examined and preserved in plastic containers. All abnormalities were recorded.

Statistics:

The following statistical methods were used:
If the variables could be assumed to follow a normal distribution, the Dunnett-Test, based on a pooled variance, was applied for the comparison between the treated groups and the control group, otherwise the Steel-Test was applied.
Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information (Bonferroni-Holm-corrected).

Historical control data:

Findings both on the individual foetus and on the litter basis did not differ from the available historical control obtained in six developmental toxicity studies on the same species.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
The dams tolerated the applied dose levels of up to 1000 mg/kg bw/day without lethality and clinical signs of systemic toxicity.
Maternal body weight gain was not affected by the treatment.

Mortality:
No death occurred in the dams of group 1 (vehicle control) and in the test groups 2 - 4.

Signs and/or symptoms:
No compound-related symptoms were observed in all treatment groups. In one female (group 3) was noted a skin incrustion on the back and another female (group 1) was severely aggressive by handling.

Body weight gains and corrected body weight:
Body weight profiles of the pregnant females were essentially similar in all groups. Mean corrected body weight gain of the treatment groups compared favourably with the control values.

Reproduction data:
No compound-related differences were noted between the mean reproduction data of the test groups in comparison to the control group. In the group 2 and 4 the post-implantation loss and total embryonic deaths were significantly decreased. These findings were considered to be incidental because of the high control values. Furthermore the number of total fetuses was increased in the group 2 and 4, which is also incidental because there was no dose-relationship.

Necropsy findings:
No macroscopic changes were noted in the dams of the groups 1 - 4.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Apart from dose group 1000 mg/kg bw/day (one dead foetus) all females had viable foetuses. Pre- and postimplantation loss and mean numbers of resorption were unaffected by treatment. All parameters were comparable with the animals of the control group. Skeletal and visceral investigations detected no treatment-related malformations.

Body weight:
The weights of live fetuses exibited no significant differences on a litter and individual basis e.g. mean weight between the control group and the treatment groups.

Placenta and uterus weight:
The weights of placentae and the whole uterus showed no significant differences between the control group and the treatment groups.

Sex ratios:
The sex ratio of the fetuses was not effected by the treatment with the test substance.

External examinations:
No substance-related macroscopical findings were noted at external examination of fetuses which were considered to be an effect of the treatment with the test article. In the group 1 was noted a beginning hydrops and in the group 4 one fetus with paleness and one dead fetus.

Visceral examination:
The findings were as follows:
Group 1: 127 examined fetuses
28 hydronephrosis
9 ureter dilatation
5 ureter waved
1 runt, brain lateral sinus dilatation, other organs normal
1 thorax - blood coagulum [artifact]
1 adrenal central pinhead cyst [suspicious]
1 umbilical region - gut protrusion [artifact]

Group 2: 138 examined fetuses
34 hydronephrosis
12 ureter dilatation
3 ureter waved
1 runt, hydrocephalus internus

Group 3: 138 examined fetuses
26 hydronephrosis
5 ureter dilatation
6 ureter waved
1 ear region subcutaneous hematoma
1 umbilical region - gut protrusion [artifact]

Group 4: 140 examined fetuses
24 hydronephrosis
10 ureter dilatation
8 ureter waved
1 inguinal hernia, protrusion of gut and testis between peritoneum and trunk, muscles [artifact]
1 runt, brain lateral sinus dilatation, other organs normal

The visceral examination of the preserved fetuses did not reveal any treatment-related abnormalities.

Skeletal examination of fetuses:
Retardations:
Group 1: 141 examined fetuses
Group 2: 152 examined fetuses
single sternebrae non ossified,
significant increase at level 1% (34 fetuses out of 22 dams)
Group 3: 150 examined fetuses: no significant findings
Group 4: 152 examined fetuses
single sternebrae non ossified,
significant increase at level 5 % (29 fetuses out of 22 dams)
two sternebrae non ossified,
significant increase at level 1 % (21 fetuses out of 22 dams)

The statistically significant differences were considered to be incidental because these retardation effects were not accompanied by weight retardation of the treatment groups. The incidental character of these retardations is emphasized by the fact the values were within the normal range of variation for this strain.

Variations (examined fetuses):
Group 1: no variations
Group 2: no variations
Group 3: no variations
Group 4: no variations

Malformations (examined fetuses):
Group 1: 1 fetus beginning hydrops
Group 2: no findings
Group 3: no findings
Group 4: no findings

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

2 -ethylhexyl stearate up to a dose of 1000 mg/kg bw/day does not produce any embryo- and foetotoxic or teratogenic effects. The NOAEL for maternal-, developmental-, embryo-, foetotoxicity and teratogenicity is deduced 1000 mg/kg bw/day.

Effect on developmental toxicity: 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, similarities in PC/ECO/TOX 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 developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity

There are no data regarding developmental toxicity available for Fatty acids, C18 unsaturated, ethyl & methyl esters. In order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex IX, 8.7, read-across to an appropriate substance is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5.

According to 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) “to avoid the need to test every substance for every endpoint”.

Fatty acids, C18 unsaturated, ethyl & methyl esters is a multi-constituent substance specified by esters of ethanol and methanol with fatty acids C18 unsaturated (source canola oil) resulting in monoesters which meets the definition of an UVCB substance. Thus, the test substance represents fatty acid esters which undergo to a high extent hydrolysis by ubiquitous expressed gastrointestinal enzymes into the free fatty acid components and the respective alcohol moieties (Lehninger, 1970; Mattson and Volpenhein, 1972). Considering the common metabolism, the read-across approach is based on the presence of a common functional group, common precursors and the likelihood of common breakdown products via biological processes, which result in structurally similar chemicals with similar physico-chemical, toxicological and toxicokinetic behaviour. For further details on the read-across approach, please refer to the analogue justification in section 13 of the technical dossier.

As no data on developmental toxicity are available for Fatty acids, C18 unsaturated, ethyl & methyl esters, read-across to reliable data on the analogue substance Fatty Acids, C16-18, 2-ethylhexyl esters (CAS 91031-48-0) was conducted. The substance was chosen based on its structural similarities regarding the common functional group and the breaking products alcohol (2-ethylhexanol) and fatty acid (linear C16-18).

CAS 91031-48-0

A prenatal developmental toxicity study was performed with Fatty Acids, C16-18, 2-ethylhexyl esters (CAS 91031-48-0) according to OECD Guideline 414 (Pittermann, 1994). Groups of 24 female Sprague-Dawley rats received daily oral gavage doses of the test substance in arachidis oil at dose levels of 0, 100, 300 and 1000 mg/kg bw/day during gestational days 6 to 15. On day 20 of gestation the animals were euthanized and examined for maternal and fetal parameters. Based on the number of implantations, number of total litter losses by resorption, mortality, clinical signs, body weight, gross pathology and organ weights of maternal animals the NOAEL for maternal toxicity was found to be 1000 mg/kg bw/day. Examination of fetus litter size and weights, offspring viability (number alive and number dead), sex ratio, grossly visible abnormalities, external, head, soft tissue and skeletal abnormalities showed no differences to control and no indication for teratogenic effects. Therefore, the NOAEL for embryo-/ foetotoxicity and teratogenicity in rats for Fatty Acids C16-18, 2-Ethylhexyl esters was found to be 1000 mg/kg bw/day.

Conclusion on developmental toxicity

Based on the available data, the analogue substance did not provide evidence to indicate that Fatty acids, C18 unsaturated, ethyl & methyl esters negatively affect organogenesis or foetal development in utero. Thus, Fatty acids, C18 unsaturated, ethyl & methyl esters is not considered to exhibit developmental toxicity.

 

A detailed reference list can be found in the CSR and in section 13 of the dossier.

Justification for selection of Effect on developmental toxicity: via oral route:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose (refer to the endpoint discussion for further details).

Justification for classification or non-classification

Based on the analogue read-across approach, the available data on reproduction and developmental toxicity does not meet the classification criteria according to Regulation (EC) 1272/2008, and is therefore conclusive but not sufficient for classification.