Methyl 12-hydroxyoctadecanoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / 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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

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.

Test material

Test material information

Constituent 1

Reference substance name:

Methyl laurate

EC Number:

203-911-3

EC Name:

Methyl laurate

Cas Number:

111-82-0

Molecular formula:

C13H26O2

IUPAC Name:

methyl laurate

Test material form:

liquid

Details on test material:

Dodecanoic acid methyl ester [methyl laurate, CAS No. 111-82-0], manufactured by Tokyo Chemical Industry Co., Ltd., Lot. No. GD 01, content 99.2%, molecular weight 214.35, melting point 5 ° C., boiling point 141 ° C., specific gravity 0.8706 (20 ° C.). Colorless and transparent liquid

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).

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.