12-hydroxy-N-(2-hydroxyethyl)octadecan-1-amide

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from J check

Data source

Reference

Reference Type:

publication

Title:

Combined Repeated Dose and Reproductive/Developmental Toxicity Screening Test of the test chemical by Oral Administration in Rats

Author:

NITE

Year:

2013

Bibliographic source:

J-check

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Combined repeated dose repro-developmental screen was performed to determine the toxic nature of test chemical.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Docosanoic acid

EC Number:

204-010-8

EC Name:

Docosanoic acid

Cas Number:

112-85-6

Molecular formula:

C22H44O2

IUPAC Name:

docosanoic acid

Details on test material:

- Name of test material: Docosanoic acid
- Molecular formula: C22H44O2
- Molecular weight: 340.59 g/mol
- Substance type: Organic
- Physical state: Solid (White powder)
- Impurities (identity and concentrations): (C14-C20) fatty acids (10.9 %) and C 24 fatty acid (2.3%)

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 8 week old
- Weight at study initiation:
Male: 312.1 to 363.7 g
Female: 205.3 to 230.8 g
- Fasting period before study: No data
- Housing: Each animal was housed individually in a conditioned room in Japan cage with wire mesh floor cage. Pregnant female rats were bred in cages with pulp and papermade chips as bedding
- Diet (e.g. ad libitum): Solid feed (CE2, Japan Clea) ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 ° C
- Humidity (%): 50 to 65%
- Air changes (per hr): 15 times / hour
- Photoperiod (hrs dark / hrs light): 12 hours (7 am to 7 pm)

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil
- Concentration in vehicle: 0, 100, 300 and 1000 mg/kg/day
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): No data available
- Purity: No data available

PREPARATION OF DOSING SOLUTIONS: Docosanoic acid was prepared by suspension in corn oil at dose levels of 0, 100, 300 and 1000 mg/kg. Dosing specimens were prepared at least once weekly

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil
- Concentration in vehicle: 0, 100, 300 and 1000 mg/kg
- Amount of vehicle (if gavage): 5 mL/Kg bw
- Lot/batch no. (if required): Lot No. V6H2050
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

Males: 43 days; Females: approx 63 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Total: 104
0 mg/kg/day: 13 males and 13 females
100 mg/kg/day: 13 males and 13 females
300 mg/kg/day: 13 males and 13 females
1000 mg/kg/day: 13 males and 13 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 14 days preliminary repeated dose study was performed. No signs of toxicity were observed even in the group administered with the critical dose of 1000 mg/kg. Hence in the main study, 1000 mg/kg was set at the highest dose, and divided by the tolerance of about 3 to make the medium dose 300 mg/kg and the low dose 100 mg/kg. Corn oil used as a medium for docosanoic acid was administered to the rats of the control group under the same conditions as the docosanoic acid administration group.

- Rationale for animal assignment (if not random): Both males and females were grouped by random weighted stratified random extraction method based on the body weight of the administration start date (8 weeks old), and 13 male and 13 female each were placed in each group.

- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked in table [No.?] were included. No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observed at least once a day.

BODY WEIGHT: Yes
- Time schedule for examinations: For males on day 1, 8 and 15 of administration and for copulated females on day 1, 8, 15, 22, 29, 36 and 42 days and post menopausal days. For non copulated females the body weight was also measured on the 22nd day of administration. Also, mating females at 0, 7, 14, and 20 days of gestation, females delivered at 0 and 4 days of nursing (autopsy days), females who did not deliver, weighed 25 days pregnant (day of autopsy).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: For males on day 1, 8 and 15 of administration and for copulated females on day 1, 8, 15, 22, 29, 36 and 42 days and post menopausal days. No food intake was measured during the 2 week mating period. For mating females, food intake was measured between 0-7, 7-14, 14-20 days of pregnancy, and 0-4 days of feeding in females delivered. In addition, feed consumption of mothers who died in the middle of nursing on 04 days of nursing were excluded from the evaluation.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY: No
- 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: Not examined

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not examined

OPHTHALMOSCOPIC EXAMINATION: Not examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of administration period.
- Anaesthetic used for blood collection: Yes, pentobarbital sodium anesthesia
- Animals fasted: No data available
- How many animals: All male animals
- Parameters checked in table [No.?] were examined:
Males: RBC, hemoglobin, hematocrit, MCV, MCH, MCHC platelet, WBC, band neurophil, segmented neurophil, eosinophile, basophile, monocyte and lymphocyte were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of administration period.
- Animals fasted: Yes, pentobarbital sodium anesthesia
- How many animals: All male animals
- Parameters checked in table [No.?] were examined.:
Males: Total protein, albumin, A/G, BUN, creatinine, glucose, total cholesterol, total bilirubin, triglyceride Na, K, Cl, Ca, inorganic phosphorus, ALP, GPT,GOT, γ-GTP and LDH were examined.

URINALYSIS: No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER:
A. Calculation of the number of births: The number of births (surviving infants + dead children) was examined on day 0 of nursing and the rate of
delivery and the birth rate of babies was obtained. The presence and sex of external birth defects in babies, and calculated the sex ratio of the survivors was also determined.

B. Calculation of the number of deceased children: The number of deceased children was checked every day and the birth rate and neonatal survival rate was noted on 4th day of feeding

C. Weight measurement: Body weight (litter weight) was measured for each sex on a 0 day and a 4 day nursing, and the average body weight per animal was obtained for each abdomen.

D. Necropsy: The surviving infant was mortalized in all the cases by ether inhalation on 4th day of nursing, and necropsied.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes,
Males: After fasting period after the end of the administration on the final administration day, necropsy was done by exsanguinating with lethality under deep anesthesia with pentobarbital sodium at the next day (day corresponding to 43 days of administration). At that time, weights of heart, liver, kidney, thymus, testis and epididymis were measured for all cases. Of these organs, the testis and epididymis were fixed and stored in Bouin's solution, and other organs and brain, spleen, adrenal gland and bladder were fixed and fixed in 10% formalin.

Females: Females delivered were sacrificed on the fourth day of nursing, females that did not deliver but did not deliver to the 25th day of pregnancy, females who did not copulate were exsanguinated / lethal under pentobarbital sodium anesthesia at the end of the mating period and autopsied. Observation of organs / tissues with the naked eyes. At the same time, the organ weights were measured for the heart, liver, kidney and thymus, and the specific body weight (relative weight) was also calculated. Ovary and uterus were excised in both cases of pregnancy and infertility, the
ovaries counted the number of pregnant lutein under a stereoscopic microscope, the number of implantation was counted for the uterus, and the implantation rate [(implantation number/pregnancy corpus luteum number) × 100] was calculated.

HISTOPATHOLOGY: Yes
Males: Organ examined: Brain, heart, liver, slpeen, thymus, kidney, urinary bladder, adrenal gland, ovary, testis and epididymis were examined. The testis and epididymis were fixed and stored in Bouin's solution, and other organs and brain, spleen, adrenal gland and bladder were fixed and fixed in 10% formalin. These organs were subjected to paraffin sections according to a conventional method for 1000 mg/Kg group and 0 mg/Kg group and subjected to histopathological examination by hematoxylin-eosin staining.

Females: The brain, heart, liver, spleen, kidney, bladder, adrenal gland, thymus, uterus and lesion were fixed in 10% formalin solution and ovaries were stored in Bouin's solution (10% formalin solution for long term storage). For all of the groups and control groups, histopathological examination of the above mentioned organs (except ovaries, uterus and lesions) was performed. However, histopathological examination was also performed on ovaries for females that had been mated but were infertile and females that did not copulate.

Other examinations:

Reproductive performance of F0 animals, development and body weight of F1 pups up to day 4 of lactation and morphological findings of F1 pups were examined.

Statistics:

For mating rate and conception rate, chi 2 test including Yates' correction was conducted. Regarding histopathological findings, one sided tests were performed on Mann-Whitney U test for grade separated data and Fisher direct probability for total positive grade values. For the other data, the value obtained for each individual or average value for each litter was set as one sample, and the variance uniformity of each group was first tested by the Bartlett method. As a result, when the variance is made uniform, a oneway type of variance analysis is performed, and if significance is found between the groups, if the number of animals in each group is the same, the Dunnett method using, if not identical Scheff were assayed for differences in mean values between the control group and docosanoic each treatment group using the method. If the variance was not uniform or there was a group where the variance was 0, Kruskal-Wallis rank test was performed, and when significance was found between the groups, the control group and docosanoic acid Dunnett method or Scheff for the difference between the administration groups was carried out method test. The level of significance was set at 5% and 1%.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Males: No change in general condition was noted in any of the treatment groups.
Females: At 100 mg/Kg, from 42nd day of dosing, piloerection, blood like discharge from the vaginal opening in one females followed by coat contamination on 43-44 day administration

Mortality:

no mortality observed

Description (incidence):

Males/Females: No abnormalities in death were noted in any of the treatment groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males: The body weight gain increased from 8 to 15 days in the group administered 100 mg / kg significantly (p<0.01) compared with the control group, and the cumulative increase from the administration start date was also significantly higher Values after 29 days increased significantly (p <0.05) than the control group. However, in the group administered with 300 mg / kg or more, there was no significant difference between the control group and body weight and weight gain at any time.

Females: No significant difference in body weight and body weight gain between control group and docosanic acid administration group before breeding, pregnancy period and nursing period was noted.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: No significant difference between the control group and docosanic acid administration group at any time of feeding intake was observed.

Females: No significant difference between the control group and docosanic acid administration group for food consumption before mating and pregnancy was observed. In the nursing stage, the consumption of 0 to 4 days of nursing during the 100 mg / kg administration group decreased significantly (p <0.01) compared with the control group, but in the group administered with 300 mg / kg or more, There was no significant difference between the control group.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males: The average red blood cell hemoglobin concentration (MCHC) decreased significantly (p <0.01) in comparison with the control group in the group administered with 300 mg / kg or more. No significant differences were found between the control group and docosanic acid administration group for other test items.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males: Alkaline phosphatase activity decreased significantly (p <0.05) in the docosanic acid administration group and in the glucose concentration 1000 mg / kg administration group, respectively, as compared with the control group. In addition, the concentration of total protein and calcium decreased significantly (p <0.05, p <0.01) compared with the control group and the chlorine concentration increased significantly (p <0.05) in the group administered with 300 mg / kg. However this change was not dose related.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males: In the 100 mg / kg administration group, the actual weight and the specific body weight value of the liver increased significantly (p <0.05) compared to the control group, but was not considered to be dose related change. Regarding other organs, no significant difference was observed between the control group and docosanic acid administration group.

Females: The actual weight of the kidney decreased significantly (p <0.05) in the 100 mg/Kg dose animals compared with the control group, but this effect was not the dose dependent change. No significant difference was observed between the control group and docosanic acid administration group in other organs.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Males/ Females: No treatment related effects due to docosanoic acid administration were noted

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Males: No significant differences were found between the control group and docosanic acid administration group for any histopathological findings.

The findings include: Myocardial degeneration of heart was observed in the animals of the control group and 1000 mg / kg administration group. Periportal lipidation of the liver was observed in the control group and 1000 mg / kg administration group, but there was no difference in frequency and degree between both groups. In addition, focal necrosis was found in one of the control group. Brown pigmentation and extramedullary hematopoiesis of spleen were observed in the control group and 1000 mg / kg administration group, but there was no difference in frequency and degree. Cortical basophilic tubules and eosinophilic bodies in kidneys were found in the control group and 1000 mg / kg group, but there was no difference in frequency and degree between the two groups. In addition, fibrosis was observed in kidneys in one of the control group. Fibrosis of the adrenal gland was observed in one of the 1000 mg / kg administration group, and there was no abnormality. Atrophy of seminiferous tubules was found in 2 animals in the 1000 mg / kg administration group, one on both sides and the other on one side, but both were localized and extremely mildly changed. There was no other abnormality. Sperm granulomas were found in the epididymis in one of the control group, and there was no abnormality. No abnormality was noted in brain, thymus and bladder.

Females: Deposition of minerals in the thalamus was noted in 1000 mg / kg administration group and no abnormality was noted. Focal necrosis was found in one of the control group and in all animals of 1000 mg/Kg group and also fibrosis was noted. Brown pigmentation and extramedullary hematopoiesis of the spleen were observed in the control group and 1000 mg / kg administration group, but there was no difference in frequency and degree between the two groups. Atrophy of the thymus and hemorrhage was observed in the control group and 1000 mg / kg administration group but no difference in control and treated animals was found. Cortical basophilic renal tubule and renal pelvic dilation of the kidneys were observed in the control group and 1000 mg/kg administration group. Necrosis was observed in one cortex in the control group, and there was no abnormality. No abnormality was noted in heart, bladder, ovaries of the unexpanded and infertile cases.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Remarks on result:

other: No effect observed

Target system / organ toxicity

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Any other information on results incl. tables

The conception rate also had no effect of docosanoic acid. There was no effect of docosanic acid administration on pregnancy period, maternal pregnancy rate, childbirth rate, labor condition and nursing condition, as well as survival, body weight, sex ratio and morphology of the infant.

Applicant's summary and conclusion

Conclusions:

The No Observed adverse effect level (NOAEL) for test chemical is considered to 1000 mg/Kg/day using male and female Crj: CD(SD) rats as animal model.

Executive summary:

Repeated dose oral toxicity study with reproductive and developmental screening was conducted to investigate the repeated dose toxicity of the test material. The chemical was administered by oral gavage route of exposure to dose groups, each of 13 male and 13 female Crj:CD (SD) strain rats upto forty-three days at dose levels of 0, 100, 300 or 1000 mg/kg/day. A control group of 13 males and 13 females was dosed with vehicle alone (corn oil). As a result, in males, abnormalities in death cases and general conditions were not observed in any treated animal. There was also no change in body weight and food consumption suggesting the effect of test chemical administration. Necropsy, histopathological examination, hematology examination, and blood biochemical examination after repeated administration of 42 times also showed no findings or abnormal values suggesting the effect of test chemical administration. In females, there were no deaths in any test chemical administration group. In addition, no change was observed in general condition, body weight and food intake. Necropsy at 4 days postpartum and histopathological examination also did not show findings suggestive of the effect of test chemical administration. On the reproductive and developmental toxicity, test chemical dose up to 1000 mg / kg did not affect mating rate and conception rate. In addition, changes suggesting the effect of test chemical administration on the pregnancy period, birth rate, labor condition and nursing condition of the mother animals were not observed. No treatment related adverse effects were found in either dose group 0, 100, 300 or 1000 mg/kg/day, though some slight changes were observed in blood biochemistry and histopathology in testes. Thus, on the basis of overall discussion of the study, the No Observed adverse effect level (NOAEL) for test chemical is considered to 1000 mg/Kg/day using male and female Crj: CD(SD) rats as animal model.