Methyl 12-hydroxyoctadecanoate

Endpoint summary

Description of key information

No adverse effects observed

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The analogue approach is used for the hazard assessment of toxicological, eco-toxicological and environmental fate endpoints for the registration of 12-hydroxstearate methyl ester (CAS 141-23- 1). The hypothesis is that data can be read-across between this ester and its structural analogues, based on structural similarity and which cause the same type of effect(s) in physical and biological systems (Scenario 2 of the Read-Across Assessment Framework (RAAF, ECHA, 2015). The primary fatty acids in this read-across are lauric acid (C12) and myristic acid (C14), as these are well studied with high-quality experimental data. Supplemental analogues are used which contribute understanding of the effects of other structural features not contained in the two primary analogues.

There are no GHS classifications for 12-hydroxstearate methyl ester for endpoints which are reliant on read-across. There is a high degree of confidence that hazards for these endpoints are not underestimated, based on a strong weight of evidence from multiple data sources.

Read-across data, all evaluated as reliable according to Klimisch scores of 1 or 2, to estimate the toxicity of the registered substance is used for fulfilling the data requirements of the REACH registration and classifying potential hazards. This read-across approach is adequate for the purposes of risk assessment and classification and labeling.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Dodecanoic acid methyl ester (abbreviation: MD, CAS No.:111-82-0, lot number: GD01, manufactured by Tokyo Chemical Industry Co., Ltd.) is a colorless transparent liquid and has a melting point of 5 ° C., a boiling point of 141 ° C. (15 mmHg) , Molecular formula C13 H26 O2, molecular weight 214.35, purity 99.2% (impurity unknown).

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crj: CD (SD), SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Crj: CD (SD), SPF rats, male and female, 9-10 week old (initiation of dosing)
- Source: Charles River Japan Co., Ltd.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9-10 wks
- Weight at study initiation: 333-411 g (males); 202-254 g (females)
- Fasting period before study: 16 h before sacrifice.
- Housing: stainless steel mesh lids, housed individually.
- Diet (e.g. ad libitum): NMF solid feed (radiation sterilized feed) manufactured by Oriental Yeast Co., Ltd., ad libitum
- Water (e.g. ad libitum): municipal supply, ad libitum.
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +/- 2
- Humidity (%): 55 +/- 10%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12, 150300 lux

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): optimal solubility
- Concentration in vehicle:
- Amount of vehicle (if gavage): 5 ml/kg bw/d
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For concentration analysis of the administration solution, samples were randomly extracted from batches of each group prepared at the start of preparation. As a result, it was prepared in the range of 98.6 to 104%, and it was confirmed that almost a predetermined amount of dodecanoic acid methyl ester was detected

Duration of treatment / exposure:

45 days (males); 44-51 days (females)

Frequency of treatment:

once daily for 7 days/wk

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 for main dose groups.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose range finding study of 100, 250, 500 and 1000 mg/kg bw/d. No adverse effects seen, and no deaths. Doses selected for main study are 250, 500, 1000 mg/kg bw/d
- Rationale for animal assignment: body weight stratified, then random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Male: 1, 8, 15, 22, 29, 36, 43, and 46 days on administration; Female: 1, 8, and 15 days on administration, 0, 7, 14, and 21 days of gestation, and days 0 and 4 of lactation
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Male: 1, 8, 15, 22, 29, 36, 43, and 45 days on administration; Female: 1, 8, and 15 days on administration, 0, 7, 14, and 21 days of gestation, and days 0 and 4 of lactation
FOOD EFFICIENCY: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes (male only)
- Time schedule for collection of blood: On the day following the end of the administration period
- Anaesthetic used for blood collection: Yes.
- Animals fasted: Yes. Over 16 h after the administration period
- How many animals: All males: 48 males (12/dose)
CLINICAL CHEMISTRY: Yes
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Organ weights: thymus, lungs, liver, kidneys, testes, epididymides, and ovaries were weighed.
HISTOPATHOLOGY: Yes
Male; Control and 1000 mg/kg bw/d groups: brain, thymus, heart, lungs, liver, kidneys, spleen, adrenal glands and testes
Pregnant female; Control and 1000 mg/kg bw/d groups: brain, thymus, heart, lungs, liver, kidneys, spleen, adrenal glands and ovaries
Non-pregnant females or infertile males; all administration groups: brain, thymus, heart, lungs, liver, kidney, spleen, adrenal glands, vagina, the uterus, the ovaries, testes, epididymides, seminal vesicle, the prostate, and pituitary gland
Dams that all children died; all administration groups: skin, mammary gland, lymph node, salivary gland, sternum, femur, thymus, lungs, bronchus, heart, thyroid gland, parathyroid, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidneys, adrenal glands, urinary bladder, ovaries, uterus, vagina, an eyeball, harderian gland, brain, pituitary gland, and spinal cord

Other examinations:

Pairing of males and females (1:1) within each treatment group on day 15, and females were allowed to deliver and rear offspring to PND 4. Reproductive parameters and indices were assessed in males and females.

Statistics:

Statistical analyses were conducted for multiple comparisons using Bartlett’s test, one way ANOVA, Dunnett’s or Scheffe’s pair wise comparison test, Kruskal-Wallis or Dunnett or Scheffe’s rank sum test, and Chi square test

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

1000 mg/kg bw/d group: statistically significant increase in daily food intake in the 250 and 500 mg / kg group on days 43 to 45 compared with the control group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Male: 1000 mg/kg bw/d group: increase in absolute weight of the thymus. Because there is no significant difference in relative weight, no pathologic findings in the thymus, and no change of the lymphocyte ratio related to hematologic inspection, there is no apparent relation with change in thymus weight and exposure

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

No deaths and no clinical signs were observed which were related to treatment with the test article. In males, the average daily food intake was increased in the 250 and 500 mg / kg group on days 43 to 45 compared with the control group. In females, there was no difference between the control group and each test substance-administered group over the administration period. Notwithstanding, body weight gain was comparable among treated groups compared to that of controls. There were no differences in test and control animals in hematology and blood biochemical examination. In males, the absolutel weight of the thymus increased in the 1000 mg / kg group (p < 0.01) compared to the control group. Because there was no significant difference in relative weight, no pathologic findings in the thymus, and no change of the lymphocyte ratio related to hematologic inspection, this was not considered an adverse/toxicological effect. No histological findings were observed between treated and control animals; there were no histological lesions seen in the thymus in high dose males. In females, there were no differences in organs between the control group and the test substance-administered group. No adverse effect of the compound was observed at any dose level on the reproductive performances, nor were there signifcant changes in gestation or developmental parameters.Pregnancies were established in all females of the 0, 500 and 1000 mg / kg dose groups, and in 250 mg / kg group, 10 out of 12 females (83.3%) were impregnated. The NOAEL(adlult) for repeated dose toxicity was 1000 mg/kg bw/d.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

An analogue substance was tested for repeated dose toxicity effects in male and female rats in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD4 TG422). The substance was administered to CD rats in a corn oil vehicle by oral gavage at doses of 0, 250, 500 and 1000 mg/kg bw/d for 42 days (males) and 42-46 days (females). A recovery group of 5 animals, high dose only, was extended for an additional 14 days. The NOAEL for repeated dose toxicity was 1000 mg/kg bwd. This data is applicable to the target substance, which is expected to behave similarly. The substance shows no local or systemic toxicity.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The analogue approach is used for the hazard assessment of toxicological, eco-toxicological and environmental fate endpoints for the registration of 12-hydroxstearate methyl ester (CAS 141-23- 1). The hypothesis is that data can be read-across between this ester and its structural analogues, based on structural similarity and which cause the same type of effect(s) in physical and biological systems (Scenario 2 of the Read-Across Assessment Framework (RAAF, ECHA, 2015). The primary fatty acids in this read-across are lauric acid (C12) and myristic acid (C14), as these are well studied with high-quality experimental data. Supplemental analogues are used which contribute understanding of the effects of other structural features not contained in the two primary analogues.

There are no GHS classifications for 12-hydroxstearate methyl ester for endpoints which are reliant on read-across. There is a high degree of confidence that hazards for these endpoints are not underestimated, based on a strong weight of evidence from multiple data sources.

Read-across data, all evaluated as reliable according to Klimisch scores of 1 or 2, to estimate the toxicity of the registered substance is used for fulfilling the data requirements of the REACH registration and classifying potential hazards. This read-across approach is adequate for the purposes of risk assessment and classification and labeling.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Qualifier:

equivalent or similar to guideline

Guideline:

other: 1993 U.S. FDA Redbook II, short term toxicity repeated dose toxicity study

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Source: Victorian Chemical, Victoria, Richmond, AU. Analytical testing from National Formulary (NF) and European Pharmacopoeia (EP) specification. High oleic safflower oil (HOSO) was obtained from Columbus Food Co., Chicago, IL, USA. Ethyl oleate (EO) fatty acids are in the form of ethyl esters.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Madison, WI, USA). Crl:CD(SD) IGS BR
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 wks
- Weight at study initiation: 150-175 g
- Fasting period before study: yes, the night prior to blood collection
- Housing: Individually in stainless steel cages
- Diet (e.g. ad libitum): AIN-93G purified, to allow addiitonal 10% of test fat, ad libitum
- Water (e.g. ad libitum): ad libitum, no other details
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: certified by independent lab to be free of contaminants, LOD 0.1 ppm

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: From: To:

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSES

Test materials were formulated into a purified diet based on AIN-93G, modified to allow incormation of an additional 10% of test fate (either ethyl oleate, High oleic safflower oil (HOSO), or a combination of the two). This involved decreasing overall carbohydrate concentration to allow the incormporation of an additional 10% of test fat without diluting out other nutrients.

Basal Diet Composition
Ingredient Percentage
Casein 24.4
L-Cystine 0.4
Corn starch 30.2
Maltodextrin 16.1
Sucrose 11.1
Soybean Oil 5.6
Cellulose 6.1
Mineral Mix 4.3
Vitamin Mix 1.2
Choline bitartrate 0.6
TBHQ 0.003
Following incorporation of 19% test fat, the final fat concentration is 15% (5% from soy bean oil). Provides 4.15 kcal/g.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability, homogeneity and concentrations in diet were analyzed and found to be stable over the course of the study. Diet concentrations were homogeneous and within 10% of target concentration.

Duration of treatment / exposure:

91

Frequency of treatment:

daily in feed

Dose / conc.:

3.3 other: %

Dose / conc.:

6.7 other: %

Dose / conc.:

10 other: %

No. of animals per sex per dose:

20 per sex per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The effects of EO were studied in rats fed with a purified diet having a 5% basal fat level, to which an additional 10% of fat can be added. These diets were calorie and fat-matched using high oleic safflower oil (HOSO) as the control fat.

- Rationale for animal assignment: random
- Section schedule rationale: random

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
Examined twice daily for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to dosing and weekly. These assessments were made outside the home cage and included changes in skin, fur, eye, mucous membranes, occurrences of secretions and excretions, changes in posture and reactivity to handling. Changes in gait were assessed weekly by allowing the animal to walk freely. Expanded clinical observations werre performed prior to treatment and weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: pre-study, on first day of treatment and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption was calculated as g food/kg body weight/day: Yes, weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes, by certified veterinary ophthalmologist using indirect ophthalmoscope and slit lamp. Eyes were dilated with mydriatic agent.
- Time schedule for examinations: once prior to treatment and once at week 13.
- Dose groups that were examined: all

HAEMATOLOGY: Yes, from jugular vein, with sodium citrate for coagulation tests and potassium EDTA for hematology tests
- Time schedule for collection of blood: following overnight fast, on days 30, 60 and 91
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: 10 selected animals/sex/group, on days 30 and 60, and in all animals at scheduled sacrifice.
- Parameters checked: red blood cell count, Hb, Hc, MCV, MCHb, MCHC, platelet count, WBC, differential blood cell count, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes, with no anticoagulant.
- Time schedule for collection of blood: days 30, 60 and 91
- Animals fasted: Yes
- How many animals:10 selected animals/sex/group, on days 30 and 60, and in all animals at scheduled sacrifice.
- Parameters checked: ALT, AST, AlkPhos, calcium, cholesterol, chloride, creatinine, γ-glutamyltransferase, glucose, globulin, BUN, total protein, sodium, potassium,inorganic phosphorus, total bilirubin, triglycerides

URINALYSIS: Yes, urine collected overnight in containers on wet ice before blood collection
- Time schedule for collection of urine: days 30, 60 and 91
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: appearance/color, bilirubin, blood, glucose, ketones pH, protein, specific gravity, urobilinogen, volume, microscopic examination of sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: prior to treatment and weekly
- Dose groups that were examined:
- Battery of functions tested:
a) Hand held and open field: reactiivity to handling, vocalization, palpebral closure, exophthalmos, excessive lacrimation, excessive salivation, respiration, appearance of fur, piloerection, muscle tone, pupillary status.
b) Elicited behaviors: auditory reactivity, proprioceptive positioning reaction, pinna response, pupillary status, pupillary respone, grip strength, nociceptive reflex.
c) Motor activity, once during week 13, in an automated photocell activity-recording device, with recording for 40 min.

IMMUNOLOGY: No

OTHER:CAGE SIDE OBSERVATIONS: Yes
- Time schedule: days 29 and 59 from animals selected for clinical chemistry, hematology and urinalysis, and from all animals on day 90.
- Cage side observations checked: Plasma ethyl oleate concentration measurements.
Procedure: 220 µl blood was collected from the jugular vein and mixed with sodium citrate solution in containers. 50 µl of the blood solution was mixed with 50 µl of a stable isotopically labeled internal standard prepared solution and 1 ml of acetone, and placed on ice. All mixing occurred within 4 min from the time of blood collection, to prevent hydrolysis of ethyl oleate substrate. Samples were frozen until analyzed, which took place using LC/MS/MS with atmospheric pressure chemical ionization (positive ion mode) using selective reaction monitoring. The method's calibration range was 0.02-10 µg/ml.

OTHER:CAGE SIDE OBSERVATIONS: Yes
- Time schedule: days 29 and 59 from animals selected for clinical chemistry, hematology and urinalysis, and from all animals on day 89. Animals were fasted overnight and feces collected from cage-pan.
- Cage side observations checked: Fecal fat. Fecal material was sifted in a fine-grade sifter to remove loose residual feed, weighed for each animal and stored in a freezer at -60 to -80 °C until analysed for total fat content using AOAC Method No. 95402.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:prior to dosing, then once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No specified

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

At sacrifice, the following organs were weighed (pairs together). Organ to body weight percentages and organ to brain weight ratios were calculated: adrenal (2), brain, epididymis (2), heart, kidney (2), liver, ovary (2), pituitary gland, prostate, spleen, testis (2), thymus, throid with parathyroid, uterus.

After 13 weeks of treatment, all surviving animals were fasted overnight, bled for clinical pathology tests and special sampling, were anesthetized with CO2, weighed exsanguinated and necropsied. Tissues were preserved in 10% neutral-buffered saline (except testes, which was preserved in Bouin's fixative). Organs from animals in the control and high-dose groups were examined histopathologically: adrenal (2), aorta, brain (cerebrum, cerebellus and medulla), cecum, cervix, colon (proximal and distal), duodenum, epididymis (2), esophagus, eye (2), femuse with bone marrose (articular surface of distal end), harderian gland, heart, ileum (with Peyer's path), jejunum, kidney (2), lacrimal gland, liver, lung with mainstem bronchi, lumph 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), sketal muscle (thigh), skin, spinal cord (cervical, thoracic, lumbar), spleen, sternum with bone marrow, stomach (nonglandular and glandular), testis (2), thymus, thyroid with parathyroid, tissues with macroscopic changes or alterations (gros lesions), tongue, trachea, urinary bladder, uterus with uterine horns, vagina, Zymbal's gland.

Other examinations:

Spermatocyte assessment: at sacrifice, males were evaluated for sperm viability. For motiling and morphology assessment, the right vas deferens was excised and immediately placed in 1% BSA in PBS at 38°C. After 3-4 min, a sample of sperm was collected using a 100 mciron cannula and loaded into a Hamilton Thorne IVOS automated sperm analyzer. 5 field/animal were selected and stored as digital images and anlyzed for percent motility. Sperm morphology of each animal was assessed with 2 slides of sperm, stained with eosin. For sperm count, the right epididymus was removed, divided in half by cross sectioning through the middle, and the tail end caudal section was frozed on dry ice and stored until analysis for total sperm count.

Estrus cycle analysis: 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 estrus cycle.

Statistics:

Control vs. treated group comparisons were evaluated at the 5.0% two-tailed probability level. If Levene's test for variance homogeneity was not significant (p> 0.05), one-way ANOVA was performed. If Levene's test was significant, ANOVA was done on rank transformed data. Post-hoc Dunnett's test was applied for control vs. treated group mean comarisons, using tranformations when necessary.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male from Gp 2 (3.3% EO) and two males from Gp 3 (6.7%) were found dead. The Gp 2 male showed signs of kidney problems; no pathology (gross or microscopic) explains the Gp 3 deaths. They were not judged by the pathologist to be related to the test material; there was no dose-response relationship.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Females in Gp. 3 (6.7%) and Gp. 4 (10%) showed lower terminal body weights and body weight gains. This was not judged to be adverse, as the weight and weight gains were within historical control ranges.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant changes in food consumption, even among females on the EO diet.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Values were minimally lower for calcium and mildly lower for inorganic phosphorus in high dose males; they were not considered adverse or toxicologically meaningful. There were no group changes to EO plasma concentrations at 30, 60 or 90 days.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Due to the decreased body weights of Gp 3 (6.7%) and Gp. 4 (10%) females, the brain to body weight ratios were slightly elevated.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Description (incidence and severity):

Functional assessments showed no effects.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Hepatocellular vacuolation typical of fat accumulation was noted for both control and high-dose animals. The incidence and severity of vacuolation were higher in animals given the control diet (fat derived from HOSO) than for Gp. 4 (10% EO) animals.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Fecal fat levels showed a dose-related increase in Gp 3 (6.7%) and Gp 4 (10%). Fecal fat in males was higher than in females. The appearance of the feces did not differ between the groups. Fertility parameters were studied in this experiment, and there were no treatment-related effects on sperm viability, motility in males nor in estrus cycle parameters in females.

Details on results:

Overall, there was no evidence of toxicity in rats consuming EO up to 10% in their diets for 90 days. Three rats from Gp 3 (6.7%) and Gp 4 (10%) died during the study, for reasons unrelated to consumption of the test material. Body weight gain was less (less than 10%) for mid and high dose females, but is not considered to be adverse, as the absolute weight and weight gains were within historical control ranges, and related to lower food consumption relative to controls. Histological examination of liver tissue showed vacuolation typical of fat accumulation for both control and high dose animals, with the incidence and severity higher in controls than the test group. Fecal fat levels increased in Gp 3 from 9 (control) to 15% in males, and from 4 (control) to 6% in females, and further increased in Gp 4 animals, to 17.5% in males and to 14% in females. There were no visual differences in feces quality or quantity. Analysis of the fat content of feces showed that 80% of the EO was absorbed, with 20% excreted in feces.

Key result

Dose descriptor:

NOAEL

Effect level:

10 other: % in diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Remarks on result:

other: approximately 6000 mg/kg bw/d

Dose descriptor:

NOEL

Effect level:

6.7 other: % in diet

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

Key result

Critical effects observed:

no

Overall, there was no evidence of toxicity in rats consuming EO up to 10% in their diets for 90 days.   Body weight gain was slightly less (less than 10%) for mid and high dose females, but is not considered to be adverse, as the absolute weight and weight gains were within historical control ranges. Weights were probably related to lower food consumption relative to controls, due to decrease palatability of the test diet compared to the control diet of triglycerides of safflower oil, which was more evident in females than males. Hepatic vacuolation was seen in control animals and the high dose Gp 4, with the incidence and severity higher in controls than the test group. This is consistent with high consumption of fat and reflects the local absorption of fat from the diet. Fecal fat levels increased in Gp 3 and Gp 4, with analysis showing significant absorption (80%) of EO, with the excess in the high EO diets eliminated in feces. Other research has shown that fatty acid esters, which are generally well absorbed in both rats and humans, may be hydrolyzed at a slower rate than typical triglycerides.  Increases in fecal absorbable fat are not associated with any known adverse health effects, with non-absorbable fat may promote the elimination of fat-soluble vitamins. There were no adverse effects in supplemental endpoints such as ophthalamological parameters, behavior, or fertility in males and females/integrity of reproductive organs. There was no evidence of toxicity associated with the hydrolzed ethyl ester (ethanol). The NOEL in this study is 6.7% EO in the diet, based on the small decreases in calcium and phosphorous in the high dose males. The NOAEL is 10% EO in the diet, equivalent to approximately 6000 mg/kg bw/d, based on the lack of any clear adverse or toxicological effects in this study.

Conclusions:

The substance was tested for repeated dose toxicity effects in male and female CD rats in a dietary 91 day repeated dose toxicity study of ethyl oleate (EO) in a purified diet (AIN 93G). Doses were 3.3%, 6.7% and 10% EO (10% approximately equivalent to 6000 mg/kg bw/d) with controls consuming diet with high oleic safflower oil (HOSO). Supplemental endpoints were added including analysis of fecal fat, ophthalamological examination, functional observational batteries, and fertility in males (sperm motility and viability) and females (estrus cycle assessment). There were no adverse or toxicological effects seen at any dose. The NOAEL for ethyl oleate for repeated dose toxicity was 10%, or 6000 mg/kg bw/d.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

6 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

adequate

System:

other: N/A

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

N/A

Additional information

An analogue substance, methyl laurate, was tested for repeated dose toxicity effects in male and female rats in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD TG422).  The substance was administered to CD rats in a corn oil vehicle by oral gavage at doses of 0, 250, 500 and 1000 mg/kg bw/d for over 28 days.   A recovery group of 5 animals, high dose only, was extended for an additional 14 days.  There was no local or systemic toxicity observed; the NOAEL for repeated dose exposure was >1000 mg/kg bwd.  This data is applicable to the target substance, methyl 12 -hydroxystearic acid, which is expected to behave similarly based on structural similarity, known metabolic pathways and common breakdown products.

A second key study is the 91-day dietary feeding study of ethyl oleate (EO) in CD rats by Bookstaff et al., 2004, at concentrations of 3,3, 6.7 and 10% in a purified diet (AIN-93G). The diets were fat- and calorie-matched, with high oleic safflower oil (HOSO) serving as the control. No significant signs of toxicity developed in exposed animals and the compound was well tolerated. The body weights of mid and high-dose females were approximately 10% lower than that of the control group, but weight gain among these groups was more than usual and was within historical control values for this rat strain. The weight differences were attributed to poor palatability based on initial decrease in food consumption, no dose-response in food consumption, lack of cumulation decrease in food consumption often observed with toxicity and anecdotal experiences with rat preference for various diets; it was not considered an adverse effect. Hepatocellular vacuolation typical of fat accumulation was present in all groups including controls, typical of livers of rats ingesting high fat diets. There was a dose-related increase in fecal fat concentration among all doses and in both sexes; but with no clinical sequela. The NOAEL was 10% EO in the diet, or approximately 6000 mg/kg bw/d.  The NOEL was determined to be 6.7% EO in the diet, approximately 3800 mg/kg bw/d, based on small decreases in calcium and phosphorus, judged not to be toxicologically significant, in the high dose males.

Additional supporting studies consistently show no systemic toxicity after repeated dose exposure.

Justification for classification or non-classification

Multiple analogues which are representative of the registered substance, when tested in repeated dose toxicity studies, display no systemic toxicity after subchronic exposure. The read-across is based on known biochemical lipid cycles and empiric evidence, and is valid for fulfilling the information requirements of this endpoint. The data does not meet the criteria for STOT-RE, according to Regulation EC No. 1272/2008.