[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

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

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:

The test substance provided by Nippon Oil & Fats (NOF) Corporation was used. The test substance was stored at room temperature until use.

Specific details on test material used for the study:

Impurities: C14-C20 fatty acid: 10.9%, C24 fatty acid: 2.3%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Hino, Tokyo
- Age at study initiation: 8 weeks
- Weight at study initiation: male: 312.1 - 363.7 g; female: 205.3 - 230.8 g
- Housing: metal wire floor cages
- Diet: ad libitum CE-2, CLEA Japan
- Water: ad libitum tap water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1
- Humidity (%): 50 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
prepared more frequently than once a week; aliquots were kept by each concentration refrigerated in airtight condition. It was confirmed that the test substance in the preparation solution was stable for 9 days in refrigeration and shading, and it was confirmed that the administered sample prepared during the test period contained a predetermined amount of the test substance uniformly.

VEHICLE
- Justification for use and choice of vehicle (if other than water): due to insolubility in water
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

- males: 42 days (from the end of mating period and the day before autopsy)
- females: from 14 days prior to mating to day 3 of lactation

Frequency of treatment:

once daily

Doses / concentrations

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

1000 mg / kg is the limit dose.
The medium dose was 300 mg / kg and the low dose was 100 mg / kg. Rats in the control group were treated with corn oil as a medium for docosanoic acid under the same conditions as in the docosanoic acid administration group.

No. of animals per sex per dose:

13 in each group

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: based on preliminary result in a 14 day-repeated dose toxicity study with the limit dose of 1000 mg / kg, where no signs of toxicity were found

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day during the breeding period

BODY WEIGHT: Yes
- Time schedule for examinations: males: days 1, 8, 15, 22, 29, 36, 42 and autopsy day; pregnancy females: premating days 1, 8, 15; pregnancy days: 0, 7, 14, 20; lactation days: 0, 4 (day of autopsy); non pregnancy females: day 1, 8, 15, 22, 25 (day of autopsy).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (males only)
- Time schedule for collection of blood: prior to autopsy
- Anaesthetic used for blood collection: Yes (identity): pentobarbital sodium (using EDTA-2K as an anticoagulant)
- Animals fasted: 18 - 24 hours before sacrifice
- How many animals: all surviving animals
- Parameters checked: red blood cell count (RBC), white blood cell count (WBC), haemoglobin content (Hb), mean corpuscular volume (MCV), haematocrit (Ht), mean corpuscular haemoglobin content (MCH), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes (males only)
- Animals fasted: 18 - 24 hours before sacrifice
- How many animals: all surviving animals
- Parameters checked: total protein, albumin, total cholesterol concentration, glucose, urea nitrogen, creatinine, alkaline phosphatase activity, GOT, GPT, γ-GTP, triglyceride concentration, inorganic phosphorus, total bilirubin, calcium, sodium, potassium, chlorine, A/G ratio

URINALYSIS: No

Sacrifice and pathology:

After deep anesthesia with sodium pentobarbital, autopsy was performed.The weights of the heart, liver, kidneys, thymus, testis and epididymis were measured.

Pathological findings and histopathology (control and 1000 mg/kg bw /day group):
male: heart, liver, spleen, kidney, adrenal, testis, epididymis, brain, thymus, bladder
female: brain, liver, spleen, thymus, kidney, adrenal, bladder, heart, ovary

Statistics:

Yates test, Mann-Whitney U test, Fisher exact test, Bartlett test, Dunnett test, Scheffe test, Kruskal-Wallis test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

significant increase was observed in 100 mg/kg bw/day group (non-adverse) compared with the control group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

significant decrease was observed in 100 mg/kg bw/day group during lactation (on 0 to 4 days of lactation) (non-adverse) for females.
In males, there was no significant difference in food intake between the control group and the docosanoic acid administration group at any time.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

significant decrease of mean corpuscular haemoglobin concentration in the 300 and 1000 mg/kg bw/day groups (non-adverse)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

non-adverse

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

non-adverse

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

non-adverse

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

non-adverse

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
- Males: No deaths or abnormalities in general condition were observed in any of the treated groups
- Females: No deaths were observed in any treated group

BODY WEIGHT AND WEIGHT GAIN
- Males: significant increase (p<0.01) compared to control was observed in 100 mg/kg bw/day group between 8 - 15 days and between 15 - 29 days. However, since no change was observed in other groups, it was considered not related to the dosing of compound.
- Females: no significant change in body weight was noted

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Males: no changes in food consumption related to the dosing of compound
- Females: significant decrease (p<0.01) compared to control was observed during lactation in 100 mg/kg bw/day group. However, since no other changes in food consumption were noted in 300 mg/kg bw/day and 1000 mg/kg bw/day, it was not related to the dosing with the compound.

HAEMATOLOGY
- Males: a significant decrease (p<0.01) of mean corpuscular haemoglobin concentration (MCHC) compared to control was found in the 300 and 1000 mg/kg bw/day dose group, respectively. No other differences were noted.

CLINICAL CHEMISTRY
- Males: a significant decreased ALP level (p<0.05) compared to control was found in all dosing groups. A significant decreased glucose level (p<0.05) compared to control was found in the high dose group. While a significant increase in chloride (p<0.05) was found in the 300 mg/kg bw/day group, a significant decrease in calcium content (p<0.01) and in total protein (p<0.05) was noted in this group. However, all the findings lack a dose-dependency and were regarded as incidental.

ORGAN WEIGHTS
- Males: in 100 mg/kg bw/day males, the actual weight ratio of liver weight compared to control values were increased (p <0.05) . No other significant differences were found in all groups. Thus, this finding lacks a dose-dependency and was regarded as incidental.
- Females: actual kidney weight was significantly reduced (p<0.05) in the 100 mg/kg bw/day group. No other weight changes were noted. Thus, this finding lacks a dose-dependency and was regarded as incidental.

GROSS PATHOLOGY
Males:
- Heart: myocardial degeneration was noted in one animal of the control group and the 1000 mg/kg bw/day group, respectively
- Liver: periportal fatty change of the liver was found in all animals of the control and the high dose group. Focal necrosis was noted in one animal of the high-dose group.
- Spleen: all animals of the control and the high-dose group showed brown pigment deposits of the spleen as well as extramedullary hematopeiosis
- Kidney: while fibrosis was only found in 1/13 animals of the control group, eosinophilic bodies and basophilic tubules in the cortex were noted in animals of both control and 1000 mg/kg bw/day groups
- Adrenal gland: one animal of the high-dose group showed fibrosis
- Testis: atrophy of the seminiferous tubules was observed in two animals of the 1000 mg/kg bw/day dose group, with one animal affected on both sides and one side affected in the other animal. No other abnormalities were noted.
- Epididymis: abnormal sperm granuloma was found in one animal of the control group
- Brain: no abnormal findings noted
- Thymus: no abnormal findings noted
- Bladder: no abnormal findings noted

Females:
- Brain: one animal of the high-dose group showed abnormal mineral deposition in the thalamus
- Liver: focal necrosis and fibrosis was found in one animal of the control group, while one animal of the high-dose group showed only focal necrosis
- Spleen: brown pigmentation and extramedullary hematopeiosis was found in animals of the control and the high-dose group, but no varying degrees of frequency was observed
- Thymus: atrophy and bleeding was observed in animals of both groups, control and high-dose group, respectively
- Kidney: basophilic tubules of the cortex and a dilatation of the renal pelvis was seen in the control and high-dose group
- Adrenal gland: cortical necrosis was observed in one animal of the control group
- Heart: no abnormal findings noted
- Bladder: no abnormal findings noted

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse substance-related systemic effects were noted

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Repeated oral administration of 100, 300 and 1000 mg / kg of docosanoic acid to male and female rats showed no deaths in any of the administration groups, and the effects of docosanoic acid administration on general condition, body weight transition and food intake. In the blood test of males after 42 doses, the mean corpuscular hemorrhage concentration was significant in the administration group of 300 mg / kg or more, the alkaline fastase activity was significant in each docosanoic acid administration group, and the glucose concentration was significant in the 1000 mg / kg administration group. However, since all the changes were mild and no concomitant changes were observed in other test items including pathological tests, these are considered to be accidental changes and have no toxicological significance. Histopathological examination showed very slight atrophy of the seminiferous tubules in 2 animals in the 1000 mg / kg administration group, both of which are spontaneous histological images due to localized changes. There was no effect of docosanoic acid on the conception rate. No effects of docosanoic acid administration were observed on maternal gestational age, fertility, parturition and lactation status, as well as birth viability, body weight, sex ratio and morphology.
Based on the above results, the no-effect level for repeated dose toxicity and reproductive and developmental toxicity of docosanoic acid is considered to be 1000 mg / kg / day under the conditions of this study.