Methyl 12-hydroxyoctadecanoate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Not a reproductive toxicant

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

Justification for study design:

The screening study is appropriate according to Annexes of Regulation EC No. 1907/2006.

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:

Details on test animals and environmental conditions
TEST ANIMALS
- (Crj: CD (SD), SPF) 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: 354 to 386 g for males and from 216 to 241 g for females
- Fasting period before study: 16 hours prior to sacrifice
- Housing: solid floor polypropylene cages with stainless steel mesh lids
. Males housed in groups; individually.
Cages were changed weekly.
- Diet (e.g. ad libitum): NMF solid feed (radiation sterilized feed) manufactured by Oriental Yeast Co., Ltd.
- 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, 150-300 lux

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test substance was dissolved in corn oil (manufactured by Nacalai Tesque, Inc.) to prepare a dosing solution for each group to have concentrations of 5, 10 and 20 (w / v)%. After preparation, it was hermetically stored under cool and dark conditions until use, and it was used within 7 days after preparation. The test substance, 5% (w / v), in the preparation solution was confirmed to be stable for at least 8 days under cool and dark conditions in the case of a solution.

Details on mating procedure:

1:1 male:female ratio
Mating confirmed by sperm presence, vaginal plug

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 ____ was contained.

Duration of treatment / exposure:

45 days (males); 41-55 days (females)

Frequency of treatment:

once daily, 7 days per week

Details on study schedule:

Body weight and food consumption were measured weekly.

Mating occured at a sex ratio of 1:1, documented by sperm confirmation in vagina and vaginal plugs. That day was taken as the 0th day of gestation.
The mating rate [(number of copulatory animals / number of living animals) × 100] and conception rate [(number of conception animals / number of copulates) × 100] were determined for each group. The sexual cycle was observed until the date of mating, and the length of the estrus cycle was calculated.

Pregnant females delivered naturally between days 20-25, and the length of pregnancy calculated. The day of delivery (nursing, lactation) was designated PND0. If labor/delivery occurred after 10 am, the following day was designated PND0. The birth rate (pregnant/delivery x 100) was calculated.

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

Control animals:

yes, concurrent vehicle

Details on study design:

Details on study design:
- Dose selection rationale:
A dose-range finding study was performed at 100, 250, 500 and 1000 mg/kg/d for 14 days. There were no observed unscheduled deaths or adverse effects on body weight, food intake, autopsy findings, organ weight, hematology values or blood biochemical values. Doses selected for the main study were 250, 500 and 1000 mg/kg bw/d.
- Rationale for animal assignment: Animals were stratified based on the body weight at the start of dosing, and 12 animals per group were sorted by a random sampling method.

Pairing of males and females (1:1) within each treatment group on day 15, and females were allowed to deliver and rear offspring to day 4 of lactation.

Reproductive parameters were assessed in males and females. Observations included clinical signs, behavioural assessments, estrous cycle assessments, body weight development and food and water consumption. Haematology and blood chemistry evaluations were made prior to mating and at the end of treatment.

Positive control:

none

Parental animals: Observations and examinations:

Observations and examinations performed and frequency
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked were included. see discussions
BODY WEIGHT: Yes
- Time schedule for examinations:
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily food consumption was calculated as g food/kg body weight/day: Yes, weekly, and for females, during gestation and during lactation
WATER CONSUMPTION AND COMPOUND INTAKE: no data
- Time schedule for examinations: no data
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: males: at end of treatment in all animals from each dose group; in females: prior to mating and at end of treatment in all dose groups
- Anaesthetic used for blood collection: yes, ether
- Animals fasted: Yes
- How many animals: all control and high dose groups.
- Parameters checked were examined. see discussions
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of the experiment
- Animals fasted: Yes
- How many animals: all control and high dose groups
- Parameters checked were examined. see discussions
URINALYSIS: no
NEUROBEHAVIOURAL EXAMINATION: no
IMMUNOLOGY: No
OTHER:CAGE SIDE OBSERVATIONS: no

Oestrous cyclicity (parental animals):

length of estrus cycle

Sperm parameters (parental animals):

none

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, births/pregnancies, pups/litter, pups/sex/litter.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, body weight PND 0 and 4, postnatal mortality (PND4), presence of gross anomalies, weight gain, physical abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

All males in each group underwent a necropsy. Animals were fasted for about 16 hours from the evening of the final administration day (administration period: 45 days) to the next morning, then opened under ether anesthesia, and blood was collected from the abdominal aorta.

Hematology examinations:
In the tests, the number of white blood cells (WBC: dark field plate method), number of red blood cells (RBC: dark field plate method), hematocrit value (HCT: dark field plate method) was calculated by using THMS H · 1 E (Calculated from RBC, MCV), hemoglobin amount (HGB: cyanomethemoglobin method), mean red blood cell volume (MCV: dark field plate method), mean red blood cell hemoglobin amount (calculated from MCH: HGB, RBC), average red blood cell hemoglobin concentration (MCHC : Calculated from HGB, HCT), platelet count (PLT: dark field plate method) and white blood cell percentage (flow cytochemistry method) were measured. The percentage of white blood cells was measured with the instrument described above, but a blood sample specimen was prepared separately and stored by May / Grunwald-Giemsa stain. Regarding the calculation of the reticulocyte (RC) ratio, a blood smear specimen was prepared after EDTA-3K added blood was stained with New Methylene Blue. Since no anemia tendency was observed in each group, no specimen was observed.

Blood clinical biochemical examination:
For the test, blood was collected in a clean seal (Yatron Co., Ltd.), and the serum obtained by standing for 30 minutes and then centrifuging at 3000 rpm for 7 minutes was analyzed using a multi-item biochemical automatic analyzer Centrifi Chem ENCORE II (Baker) and EKTACHEM 700N Total protein (Buret method), albumin (BCG method), A / G (calculated value), blood sugar (glucose oxidase method), total cholesterol (cholesterol oxidase method), urea nitrogen (urease ammonium indicator ), Creatinine (Jaff method), total bilirubin (diazo method), glutamic acid oxaloacetate transaminase (IFCC method), glutamic pyruvic acid transaminase (IFCC method), γ-glutamyl transpeptidase (Orl wski method), potassium (electrode method) , Chlorine (electrode method), calcium (arsenazo III method) and inorganic phosphorus (molybdic acid blue method) were measured.

Pathology examination:
Necropsy and organ weights
(1) male animals:
From the evening on the day of administration for 45 days, after fasting for about 16 hours, animals were exsanguinated under ether anesthesia and euthanized. At necropsy, macroscopic observation of the main organs was performed and the weights of the thymus, lung, liver, kidney, testis and epididymis were measured to calculate organ weight / body weight ratio (relative weight). Also, skin was fixed with 10% neutral buffered formalin solution as organs / tissues in which change was observed as brain, heart, spleen, adrenal gland, seminal vesicle, prostate, pituitary gland and macroscopic findings in addition to weight measurement organ of all animals did. The testis and epididymis were fixed with Bouin's solution.
(2) females spontaneously delivered:
On PND4, animals were exsanguinated and euthanized by ether anesthesia. At necropsy, macroscopic observation of the main organs was performed, and then the thymus, lung, liver, kidney and ovary weight were measured to calculate organ weight / body weight ratio (relative weight). The large intestine was also fixed with 10% neutral buffered formalin solution as organs / tissues in which changes were observed as brain, heart, spleen, adrenal gland, pituitary gland and macroscopic findings in addition to weight measuring instruments of all animals. At the time of necropsy, the number of corpus luteum and the number of implantation traces were examined, and the implantation rate [(number of implantation / number of pregnant corpus luteum) × 100] was obtained.
(3) females not undergoing natural births:
On GD 25, animals were exsanguinated and euthanized by ether anesthesia. At necropsy, after macroscopic observation of the main organs, macroscopic observation of the main organs was carried out, and the skin, mammary gland, lymph node, salivary gland, sternum, femur (including bone marrow), thymus, trachea, lung and bronchus, heart, thyroid and parathyroid, The esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenal gland, bladder, ovary, uterus, vagina, eyeball, Harder's gland, brain, pituitary and spinal cord were fixed with 10% neutral buffered formalin . Animals that did not show implantation marks were judged to be infertile.
(4) Females who delivered all stillborns:
On the day when all the surviving offspring were confirmed dead or were sacrificed, animals were euthanized after exsanguination under ether anesthesia. At necropsy, after macroscopic observation of the main organs, macroscopic observation of the main organs was carried out, and the skin, mammary gland, lymph node, salivary gland, sternum, femur (including bone marrow), thymus, trachea, lung and bronchus, heart, thyroid and parathyroid, The esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenal gland, bladder, ovary, uterus, vagina, eyeball, Harder's gland, brain, pituitary and spinal cord were fixed with 10% neutral buffered formalin . At necropsy, the number of corpus luteum and the number of implantation traces were examined, and the implantation rate [(number of implantation / number of pregnant corpus luteum) × 100] was determined.

Histopathology examination
(1) Males who successfully mated: brain, thymus, heart, lung, liver, kidney, spleen, adrenal gland and testis of all control and high dose groups.
(2) females spontaneously delivered :Brain, thymus, heart, lung, liver, kidney, spleen, adrenal gland and ovary of the control group and all the high dose groups.
(3) males who did not successfully mate and females who failed to become pregnant: on all animals, brain, thymus, heart, lung, liver, kidney, spleen, adrenal gland, vagina, uterus, ovary, testis, epididymis, seminal vesicle, prostate and pituitary.
(4) Females who delivered all stillborns: mammary gland, lymph node, salivary gland, sternum, femur (including bone marrow), thymus, trachea, lung and bronchus, heart, thyroid and parathyroid, tongue, esophagus, stomach, duodenum, small intestine, large intestine, Liver, pancreas, spleen, kidney, adrenal gland, bladder, ovary, uterus, vagina, eyeball, Harderian gland, brain, pituitary and spinal cord.

Statistics:

Data was assessed by multiple comparison methods 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. For birth rate, mating rate and conception rate, Chi square tests were used. Fisher's probability test method was used for the abnormality frequency. For the results on newborns during the post-natal period, the average per mother (litter) was taken as one sample. The levels of significance for p < 0.05 and p < 0.01.

Reproductive indices:

Corpora lutea, implantation sites, number of births, number of stillbirths, estrus cycle length, length of gestation, matings/pregnancies

Offspring viability indices:

sex ratio, average sex, delivery rate, birth rate, outlier abnormality incidence rate, survival on PND4

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):

no effects observed

Description (incidence and severity):

In males, the average daily food intake was high in the 250 and 500 mg / kg group on the 43 to 45 days of administration 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.

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effects were seen on estrus cycle parameters or fertility.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No effects were seen on sperm viability or motility parameters or fertility.

Reproductive performance:

no effects observed

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(maternal toxicity) 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

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Abnormalities were not observed in the reproductive parameters, specifically the incidence of pregnancies, the pregnancy duration of each group, the number of corpora lutea, the number of implantation sites, the number of live births and the number of stillborns, the birth rate, the implantation rate, and deliveries. There were no differences between the groups in the rate, birth rate and survival rate on PND4. There was a difference in the sex ratio, with a higher number of females born to control animals. The change in the control value contributed to a statistically significant decrease in male: female ratio in the high dose group on PND 0 and 4. in comparison with the hsitorical control value (0.84 to 1.08), it was considered that the value was accidentally high, and it could not be considered to be a toxicologically meaningful change. There were no abnormal findings in the gross pathology examination of offspring. The NOAEL(F1) was 1000 mg / kg / day.

Key result

Dose descriptor:

NOAEL

Generation:

F1

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

Key result

Reproductive effects observed:

no

Table: Summary of reproductive/developmental toxicity/offspring

Dose (mg/kg bw/day)	0	250	500	1000
Number of pregnant females	12	10	12	12
Number of pregnant females with live pups	12	10	12	12
Gestation index (%)	100.0	100.0	100.0	100.0
Gestation length in days	22.9 ± 0.5	22.6 ± 0.5	22.6 ± 0.5	22.4 ± 0.5
Number of corpora lutea	18.4 ± 4.2	16.8 ± 1.5	17.0 ± 2.7	17.4 ± 2.2
Number of implantation sites	15.7 ± 4.2	16.1 ± 1.4	14.4 ± 3.2	15.0 ± 2.6
Implantation index	84.0 ± 19.3	95.9 ± 4.9	85.2 ± 19.2	86.7 ± 15.5
Day 0 of lactation
Number of pups born	14.3 ± 4.2	14.7 ± 2.2	13.1 ± 3.7	13.6 ± 3.1
Delivery index(%)	89.8 ± 12.6	91.2 ± 9.4	88.9 ± 12.7	90.6 ± 13.0
Number of live pups	14.2 ± 4.1	14.6 ± 2.0	13.0 ± 3.6	13.6 ± 3.1
Male	8.3 ± 3.0	8.1 ± 2.8	6.1 ± 2.2	5.5 ± 2.1
Female	5.8 ± 2.3	6.5 ± 1.8	6.9 ± 2.9	8.1 ± 3.0
Sex ratio (male/female)	1.58 ± 0.79	1.41 ± 0.71	0.96 ± 0.60 (11)	0.79 ± 0.46*
Live birth index (%)	99.5 ± 1.6	99.4±1.8	99.8 ± 1.8	100 ± 0
Number of dead pups born
stillbirth	0.0±0.0	0.1±0.3	0.1±0.3	0.0±0.0
cannibalism	0.1±0.3	0.0±0.0	0.0±0.0	0.0±0.0
Pups weight (g)
Male	6.1 ± 0.6	6.2 ± 0.5	6.7 ± 0.7	6.5 ± 0.6
Female	5.8 ± 0.6	5.8 ± 0.6	6.2 ± 0.5	6.1 ± 0.6
Day 4 of lactation
Number of live pups
Male	7.0±4.0	7.2 ± 2.7	5.9 ± 2.1	5.3 ± 2.3
Female	4.8 ± 2.7	6.1 ± 1.7	7.4 ± 2.0	8.1 ± 3.0*
Viability index (%)
Male	76.7 ± 39.9	91.3 ± 20.0	97.8 ± 5.2	95.1 ± 11.5
Female	78.2 ± 37.8	94.5 ± 9.6	97.5 ± 5.8	100 ± 0.0
Pups weight (g)
Male	8.8 ± 1.4	7.9 ± 1.4	9.4 ± 1.4	9.2 ± 1.3
Female	8.5 ± 1.3	7.6 ± 1.3	8.8 ± 1.2	8.8 ± 1.3

Sex ratio (male/female) on day 4 (%)

Parenthesis indicates the number of litters evaluated.

*: significant difference from control, p<0.05

Gestation index (%) = (Number of females with live pups/Number of pregnant females) x 100

Implantation index (%) = (Number of implantation sites/Number of corpora lutea) x 100

Deliver index (%) = (Number of pups born/Number of implantation sites) x 100

Live birth index (%) = (Number of live pups on day 0/Number of pups born) x 100

Viability index (%) = (Number of live pups on day 4/Number of live pups on day 0) x 100

Conclusions:

In the reproductive toxicity portion of an OECD 422 Combined repeated dose toxicity with reproductive toxicity screening test in CD rats at doses of 250, 500 and 1000 mg/kg bw/d, the test substance did not show evidence of maternal toxicity or toxicity to offspring. The NOAEL(maternal toxicity) was > 1000 mg/kg bw/d, and the NOAEL(F1) was > 1000 mg/kg bw/d. There is no evidence for classification for reproductive hazard according to Regulation EC No. 1272/2008.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

adequate

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Quality of whole database:

adequate

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Quality of whole database:

adequate

Effects on developmental toxicity

Description of key information

Not fetotoxic or teratogenic

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Remarks:

Additionally under GV-SOLAS (Animal Welfare Legislation and Guidelines issues by the Society of Laboratory Animal Science)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS: animals were supplied at day 0 (GD0) of gestation, confirmed by vaginal plug.
- Source: Charles River, Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 8, weeks
- Weight at study initiation: 195.9-198.6 g
- Fasting period before study: no data
- Housing: semi-barrier sustained animal room, individually in Markrolon type M3 cages
- Diet (e.g. ad libitum): Altromin No. 1324, Altromin GmbH, Lage, Germany, ad libitum
- Water (e.g. ad libitum): community tap water, ad libitum
- Acclimation period: 5 days

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

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

other: arachidis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): daily
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): optimal solubility
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required): arachidis oil, DAB 10
- Purity:PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

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

Details on mating procedure:

No mating in the experiment; animals were supplied at GD0 of gestation, confirmed by vaginal plug.

Duration of treatment / exposure:

GD 6-15

Frequency of treatment:

once daily

Duration of test:

20 days

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were randomly assigned to test groups using random tables. They were treated and euthanised (ether) on GD20 in accordance with Animal Welfare legislation and guidelines. Foetuses were removed by Caesarian section.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: GD0, 6, 16 and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20 by ether overdose. Foetuses removed by caesarean section.
- Organs examined: Gross macroscopic examination of all maternal organs according to OECD 414 requirements, with emphasis on uterus, uterine contents,and record of the number of corpora lutea noted. The number and distribution of intrauterine implantations were recorded. The pre- and post-implantation loss and intrauterine resorption sites were evaluated. Intrauterine deaths were classified on the basis on absence (early resorption) and of the presence (late resorption) of foetal or decidual tissue in addition to placental tissue. Living and dead foetuses were sexed, weighed individually with placentaie and examined for gross extrernal abnormalities.

OTHER:

Ovaries and uterine content:

Examination was made of each animal's uterus, uterine contents, position of the foetuses and presence of corpora lutea, and pre- and post-implantations. The pre-implantation and post-implantation loss and intrauterine resorption sites were evaluated. Intrauterine deaths were classified on the basis of the absence (early resorption) and of the presence (late resorption) of foetal or decidual tissue in addition to placental tissue.

Fetal examinations:

Living and dead foetuses were removed from the uterus. The living foetuses were sexed, weighed individually with placentae and examined for gross external abnormalities. Foetal soft tissue and skeletal development were investigated. The Wilson technique was applied to half of the foetuses to evaluate potential visceral changes (Wilson and Warkany, 1965). The remaining foetuses were cleared in a solution of potassium hydroxide and stained with Alizarin Red according to Dawson (1926). Alterations in foetuses were classified according into four categories (Klimisch et al., I992): variations, retardations, anomaly and malformations.

Statistics:

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. The Steel test was applied when the data could not be assumed to follow a normal distribution. Fisher's exact test for 2 x 2 tables was applied if the variables could be dichotomized without loss of information (Bonferroni - Holm corrected).

Indices:

not specified but data provided in tables

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

2-Ethylhexyl stearate did not cause adverse effect to the maternal rats at any time during pregnancy. No mortalities occurred in the dams during the study, either in the vehicle control or in the groups exposed to 2-ethyl hexyl stearate up to 1000 mg/kg body weight. The absolute and the corrected body weight (Table 2) and body weight gain was comparable between the groups. No deviation was observed during the treatment period. Gross macroscopic examination of the maternal organs including ovaries and uterus revealed no alterations. The no-adverse-effect dose of 1000 mg/ kg body weight corresponds to the NOAEL of the repeated dose toxicity data in rats.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus was found dead in the 1000 mg/kg bw/d group; this was not different from the rate in concurrent controls or historical controls.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

One foetus in the control group had hydrops.

Visceral malformations:

no effects observed

Description (incidence and severity):

One foetus in the 100 mg/kg bw/d group had hydrocelphalus internus; not different from controls. The rate of hydronephrosis in treated fetuses was comparable to that of controls.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

The weights of live foetuses exhibited no significant differences on a litter and individual basis. In comparison with the control group in the 100 and 1000 mg/ kg body weight group, the post-implantation loss and total embryonic deaths were significantly decreased. Non-dose-related findings were considered to be incidental, compared to control values. Mean foetal placental and uterus weights were not affected by treatment. Foetal sex ratio was comparable in all groups. A hydrops was noted in one control group foetus.

Incidences were also compared to the historical controls of the OECD guideline No. 414 studies using the same strain (Sprague-Dawley CD). Findings both on the individual foetus and on the litter basis did not differ from the historical control. From the visceral examinations, one hydrocephalus internus was found which has to be considered as an individual non-dose-related finding which also occurs in the historical data at a comparable level (0.4 %). All other changes were slight and also occurred in the control population. Concerning the skeletal examinations on the basis of the foetal incidence, there are differentiated results in some findings such as 'non-ossification' of sternebrae (retardation). However, the total frequency of the findings ' incomplete ossification' including 'non-ossification' of all sternebrae show no substantial differences between the untreated and treated groups.

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

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In a guideline OECD 414 developmental toxicity study of 2-ethylhexyl stearate in CD rats at 100, 300 and 1000 mg/kg bw/d in arachidis oil, no adverse maternal effects, fetotoxic effects or teratogenic effects or variations were observed. The NOAEL for maternal toxicity is > 1000 mg/kg bw/d, and the NOAEL for developmental toxicity is > 1000 mg/kg bw/d. The substance is not classified for developmental effects according to Regulation EC No. 1272/2008.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

144

Species:

rat

Quality of whole database:

adequate

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Quality of whole database:

adequate

Justification for classification or non-classification

There is no experimental evidence that analogues including fatty acid esters, which encompasses the registered substance, are reproductive toxicants or teratogens. The read-across is based on known biochemical lipid cycles and empiric evidence for target and source substances, and is valid for fulfilling the information requirements of this endpoint. The criteria for classification according to Regulation EC No. 1272/2008 is not met, and the substance is not classified for reproductive toxicity.