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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
other: read-across from supporting substance (analogue or surrogate)
Adequacy of study:
key study
Study period:
2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
only Japanese translation available

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

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)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Test material form:
solid
Details on test material:
at room temperature appears as a yellow waxy paste
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Purity > 99.9 %

STABILITY AND STORAGE CONDITIONS:
- Storage condition of test material: stored under cool and dark condition (2 to 6°C)
- Storage condition of vehicle: corn oil stored in a cold place (2-6 °C), tightly closed under lighy shielding until use

It was confirmid that the test substance under storage conditions and dosage form was stable

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 (Kanagawa)
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 10 weeks
- Weight at study initiation: The mean body weight for male 372 (352 to 426) g and for female 224 (192 to 249) g
- Housing: wire mesh cage and raised with solid feed (Lab MR Stock, Nippon Nogyo Sangyo). Females after mating confirmation were placed in a polycarbonate cage containing nest-making material (White Flake, Japan Charles River).
- Diet: ad libitum
- Water: ad libitum
- Recovery period: 14 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1°C - 23.2°C
- Humidity (%): 48-61%

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Method of administration:using a syringe fitted with Teflon stomach sonde, once a day (in the morning), 14 days before the start of the mating, females from 4 days after parturition. Males and the female satellite group were orally administered for 42 days. Corn oil was similarly administered to the control group.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosage of the test sustance is prepare as a solution with a concentration to give prescribed dose by using solvent oil (Nacalai Tesque) as a solvent

DIET PREPARATION
- Type of food: solid food

VEHICLE
- Justification for use and choice of vehicle (if other than water): 9-cis-octadecenoic acid (2,3-dihydroxypropyl) ester is insoluble in water but is soluble in food oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Males: for 42 days, starting two weeks before mating.
Females: starting two weeks before mating, during mating, pregnancy and until 4 days post-partum
Frequency of treatment:
daily, in the morning
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw (total dose)
Remarks:
study dose
Dose / conc.:
300 mg/kg bw (total dose)
Remarks:
study dose
Dose / conc.:
100 mg/kg bw (total dose)
Remarks:
study dose
Dose / conc.:
0 mg/kg bw (total dose)
Remarks:
study dose
No. of animals per sex per dose:
5 animals for each group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As a dose setting test, the test substance was repeatedly administered orally at doses of 0, 100, 300, and 1000 mg / kg for 14 days to 5 rats of each group. Weight and food consumption measurements, hematology and blood biochemical examination, necropsy and measurement of organ weights were carried out. As results, toxic effects due to administration were not observed. Therefore, the dose in this study was 1000 mg / kg / day as the highest dose, then 3 doses of 300 and 100 mg / kg / day.
- Post-exposure recovery period: was set in 14 days.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On the day before the start of administration and then once a week
- Parameters checked: ease of withdrawal from the cage, ease of handling when exiting the cage, body tension (relaxation - tonic), skin (color), coat, piloerection, ocular secretion, eyelid closure state, eyes projection, lacrimation, mouth nose stains (dirt), salivation, urine contamination of the lower abdomen coat, stains around the anus, vocalization, respiration, posture, convulsions, tremor, search behavior (arousal level), alertness, locomotor activity abnormal behaviors (self bite, retrograde walking, etc.), stereotyped (excessive hair repair, repeated swiveling motion, etc.), consciousness failure (confusion, catalepsy, coma), limb muscle tone degree, urination, and excretion.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week and on the date of slaughter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The consumption of food was measured once a day for 24 hours on a daily basis.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 5pm (during administration period and the day after the completion of the recovery perid)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- Parameters checked: red blood cells count, hemoglobin amount, hematocrit, MCV, MCH,MCHC, white blood cells count, platelet count prothrombin time, total protein, albumin, A/G ratio, glucose, triglyceride, total cholesterol, total bilirubin, urea nitrogen, creatinine, AST,
ALT, g-GTP, ALP, LDH, cholinesterase, calcium and inorganic phosphorus, sodium, potassium and chlorine

URINALYSIS: Yes /
- Metabolism cages used for collection of urine: Yes
Animals were housed in a metabolism cage for 40 days (males) and on 8 days on recovery for satellites.
- Parameters checked: observation of appearance, Qualitative inspection of pH, occult blood, protein, sugar, ketone bodies, bilirubin, urobilinogen and examination of sediment. Further, for urine obtained by storing for 18 hours, urine volume, specific gravity and sodium and potassium.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examamination: Males were administered 41 days after females, once during feeding period, and on the final administration day and recovery day 13 for males and females of the satellite group
- Battery of functions tested: sensory activity / grip strength / motor activity / other: the landing foot width, grip strength of forelimb and hindlimb, spontaneous momentum ( males for 30 minutes and 60 minutes, females for 30 minutes). Spontaneous momentum of female of satellite group was also measured for 60 minutes. Males in the final administration day, females once during the feeding period, males and females of the satellite group were examined about receive auditory reaction, visual reaction, tactile reaction, ear response Injection, pain sensation response, pupillary reflex, ipsilateral flexor reflex, eyelid reflex and right flank reflex.
Sacrifice and pathology:
pathological examination:
Male planned slaughtered animals were delivered the next day on the 42nd day of administration, cases where the female was delivered well and the nursing was also smooth on the 5th day of the nursing, the case where the mating was not established was 24 days after the mating period.
Blood was sacrificed under anesthesia, and the body surface, opening mucosa and internal organs were observed macroscopically. In addition, the liver, kidney, adrenal gland, thymus, spleen, brain, heart were observed.
The experiment was conducted on these preserved organs. All the cases were examined for macroscopic abnormalities in each group. In the examination, paraffin sections were prepared according to a conventional method and subjected to microscopic examination with HE staining.
Formation cycle tests (stages II · III, V, VII and XII) were also performed.
Statistics:
Regarding the average value or frequency obtained, a significant difference (a risk ratio of 5% or less) from the control group was confirmed for quantitative data and parametric data when the test group had three or more groups , Bartlett's variance test was carried out, and if the variance was uniform, a one-way analysis of variance was performed. If the variance is not uniform and nonparametric data, We ranked Kruskal-Wallis's ranking. When significant differences were found as a result of these results, multiple comparisons were carried out by the Dunnett or Dunnett type test method. The test group consisted of 2 groups In case of parametric data, F test is carried out, and if the variance is uniform as a result, Student's t test is made, and if the variance is not uniform, Aspin-Welch's t test went. Also, nonparametric data was Mann - Whitney 's U test. Categorical data was tested using Fisher's direct stochastic method.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No change due to administration of the test substance was observed during the administration period and during the recovery period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
A small tumor was confirmed in the lower abdomen after 40 days of administration in one female of the 300 mg / kg group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no significant change in body weight and weight gain during the administration period. (results in Fig. 1; 2.1 and 2.2 of the attachment)
During the recovery period, body weight gain of a male in the 1000 mg / kg group showed a significant high value, but this was due to the low value trend of weight gain of the control group rather.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No change due to administration of the test substance was observed during the administration period and during the recovery period.
There was no significant change in body weight and weight gain during the administration period.
During the recovery period, the body weight gain of the male in the 1000 mg / kg group showed a significant high value, but this was due to the low value trend of weight gain of the control group rather.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No change due to administration of the test substance was observed. (results in table 2 of the attachment)
There was a somewhat lower value overall compared to the control group, and a significant difference was observed in the 100 mg / kg group and the 300 mg / kg group, but no dose correlation was observed in the change.
In the examination at the end of the recovery period, no significant change was observed in each examination item.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period, significant levels of inorganic phosphorus were observed in females at 300 mg / kg group and 1000 mg / kg group. These values correspond to the control group. Although dose correlation was not clear. (results in table 3 of the attachment)
In the examination at the end of the recovery period female cholinesterase in the 1000 mg / kg group showed a significant high value.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No significant changes were observed in the study during the administration period and during the recovery period. (results in table 1 of the attachement)
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No significant change was observed in each examination item in the examination at 6 weeks of administration and 2 weeks of recovery.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period, significant lower values of seminal vesicle absolute weight, 100 mg / kg females and spleen relative weights were significantly lower in males in the 100 mg / kg group. However it was not a dose-correlative change. .
At the end of the recovery period, significant lower values of relative weight were observed in the pituitary in males and in thyroid in females in the 1000 mg / kg group. (results in table 4)
Gross pathological findings:
not examined
Neuropathological findings:
no effects observed
Description (incidence and severity):
No significant change was observed in each examination item in the examination at 6 weeks of administration and 2 weeks of recovery
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No change due to administration of the test substance was observed. (results in tables 5, 6 of the attachement)
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No change due to administration of the test substance was observed.
Other effects:
no effects observed
Details on results:
In the hematological examination, a significant low value of activated partial thromboplastin time (APTT) was observed in males of 100 mg / kg group and 300 mg / kg group, but in the background data The change in the normal range (17.1 to 25.0 sec), dose correlation was not observed. In the blood biochemical tests, females of 300 mg / kg group and 1000 mg / kg group received inorganic phosphorus , But this also changed in the normal range (6.6 to 9.6 mg / dL), no other related changes were observed, and dose correlation was not clear either. Therefore, changes in these hematology tests and blood biochemical tests were judged as findings unrelated to administration of the test substance. In the organ weight, 100 mg / kg
In the group, there was a significant low value of only the absolute weight of the seminal vesicle in males, a significant low value only in the relative weight of the spleen in females, but neither dose correlated only change , And since histological examination also showed no change in these organs, it was considered as a finding unrelated to administration of the test substance. In histopathological examination, No change due to the administration of the test substance was observed in any of the organs. Regarding fibroadenoma of the mammary gland observed in one female of the 300 mg / kg group, in female rats Benign tumors 2, 3, 4 with relatively high incidence of expression, and it is judged to be spontaneous. On the other hand, in the satellite group, males had lower values of pituitary relative weight, females had thyroid The low value of the relative gland weight and the high value of the locomotor activity and cholinesterase were observed. However, during the period of administration and at the end of the administration period, Since neither change tendency is observed nor any of them is a mild change, it does not suggest a delayed toxic effect, and they are also unrelated to administration.

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:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
neuropathology
organ weights and organ / body weight ratios
urinalysis

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Any other information on results incl. tables

no significant effects related to the administration were observed at the highest tested dose.

NOAEL = 1000 mg/kg/day or more in both males and females

details about observations are indicated in the tables attached as background materials.

Applicant's summary and conclusion

Conclusions:
NOAEL 1000 mg/kg/day or more in both males and females, because this is the highest dose tested.
Executive summary:

In the present study the test substance did not induce any treatment related deaths.

No change due to the administration of the test substance was observed in any of the organs examinated.

The NOAEL was determined to be 1000 mg/kg/day or more in both males and females, because this is the highest dose tested.