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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): glycerol oleate
- Physical state: light yellow pellet
- Analytical purity: 99.93%
- Stability under test conditions: verified
- Storage condition of test material: in the dark and cool

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: 352-426 g (males; mean: 372 g), 192-249 g (females; mean: 224 g)
- Housing: individual in stainless steel cages
- Diet: ad libtum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1 - 23.2
- Humidity (%): 48 - 61
- Air changes (per hr): more than 10
- 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:
The test substance was diluted in appropritate amounts of corn oil for each dose level. Aliquots of the dosing solution corresponding to the amount of daily administration were stored in the dark at 2 - 6 °C. The stability of the dosing solution was 7 days in a refrigerator and 1 day at room temperature. Therefore, the dosing solution was used within 7 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance showed low solubility in water.
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): V4N3566
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No details reported.
Duration of treatment / exposure:
males: 42 days (14 days prior to mating and 28 days thereafter)
females: 42-52 days (from 14 days before mating to day 4 of lactation)
satellite males and females: 42 days and 14 days post-exposure observation period
Frequency of treatment:
once daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 females in control and test groups
7 males in control and 1000 mg/kg bw groups
12 males in 100 and 300 mg/kg bw groups
5 animals per sex in satellite control and test groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Two dose range finding studies were performed. 2000 mg/kg bw of test substance was administered for 3 days in male and female rats. No abnormalities were found in general condition and body weight. The second study was performed at dose levels of 0, 330, 100, 300 and 1000 mg/kg bw/day for 14 days. No abnormalities of general condition, body weight, food consumption, hematological findings, blood biochemical findings, gross pathology and organ weight were found. Therefore, 1000 mg/kg bw/day was selected as the highest dose.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first exposure and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: Day 1 (before administration), 7, 14, 21, 28, 35 and 42 (before sacrifice)
Females: Day 1 (before administration), 7, 14, during pregnancy on Day 0, 7 14 and 21, during lactation on Days 0 and 4 (before sacrifice)
Satellite males and females: Day 1 (before administration), 7, 14, 21, 28, 35, 42, 49 (Day 7 of recovery period) and 56 (Day 14 of recovery period, before sacrifice)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after last application
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters checked: RBC, Hemoglobin, Hematocrit, MCV, MCH, MCHC, Platelets, Reticulocytes, PT, APTT, WBC, Differential leukocytes: Lymphocytes, Neutrophils, Eosinophils, Basophils, Monocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after last application
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters checked: AST, ALT, ALP, γ-GTP, T-protein, Albumin, A/G, T-bilirubin, BUN, Creatinine, Glucose, T-cholesterol, Triglyceride, Na, K, Cl, Ca, P, LDH, choline esterase

URINALYSIS: Yes (males)
- Time schedule for collection of urine: Day 37 during administration period or Day 9 during recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Colour, cloudiness, water consumed, volume, specific gravity, Na, K, pH, Protein, Glucose, Ketone body, Bilirubin, Occult blood, Urobilinogen, Epithelial cells, Erythrocytes, Leukocytes, Casts, Crystals, Fat

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Males: on Week 6 of the administration period
Females: on Week 6 of the administration period
Males and females of satellite groups: on Week 6 of the administration period and on Week 2 of the recovery period
- Dose groups that were examined: all
- Battery of functions tested: hearing reaction, eye sight reaction, sense of touch reaction, pain reaction, pupil reflex, pinna reflex, ipsilateral flexor reaction, eyelid reflex, righting reflex
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Body surface, mucous membranes and internal organs

HISTOPATHOLOGY: Yes
Brain, pituitary, thyroid, thymus, lung trachea (after liquid immersion fixation), stomach, intestines, heart, liver, spleen, kidney, adrenal gland, bladder, testis, epididymis, prostate, seminal vesicles, ovaries, uterus, spinal cord (cervical, thoracic, lumbar), sciatic nerve, bone marrow (femur), lymph nodes (cervical lymph node, mesenteric lymph nodes), mammary gland, and other gross abnormalities
Spermatogenic cycle (Stage II, III, V, VII, and XII) was also investigated.

SACRIFICE
- Males: on Day 43
- Females: on Day 5 of lactation
- Females (unsuccessful mating): on Day 53
- Females (mated but non-pregnant): on Day 5 after scheduled delivery
- Satellite males and females: on Day 15 of the recovery period
Other examinations:
Organ weight: brain, liver, kidney, spleen, heart, thymus, thyroid, pituitary, adrenal, testis, sminal vesicle, epididymis
Estrous cycle
Number of ovarian corpora lutea
Number of uterine implantation
Observation of pups
Statistics:
ANOVA, Barlettm Kruskal-Willis, Dunnett, F test, Studen t-test, Aspin-Welch t-test, Mann-Whitney U-test, Fisher's exact 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):
1000 mg/kg bw/day (satellite group, males): significant increase was observed (non adverse)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day (satellite group, females): significant increase on Day 14 of administration (non adverse)
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):
100 and 300 mg/kg bw/day (males): decrease in APTT without dose-dependency after administration period (non adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
300 and 1000 mg/kg bw/day (females): significant increase in organic P in females after administration period without dose-dependency (non adverse); 1000 mg/kg bw/day (satellite group, females): significant increase in choline esterase (non adverse)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw/day: significant decrease in absolute weight of seminal vesicles in male and significant decrease in relative weight of spleen in females after administration period (non adverse)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/day: mass in subcutis in one female after administration period (not treatment-related)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
control and 1000 mg/kg bw/day: after administration period, low incidence of commonly found microscopic changes, evenly distributed between groups (not treatment-related)
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/day: after administration period, benign fibroadenoma of the mammary gland in one female (not treatment-related)
Details on results:
CLINICAL SIGNS AND MORTALITY
No abnormalities and no mortality were observed.

BODY WEIGHT AND WEIGHT GAIN
No change of body weight and weight gain was observed during the administration period. During the recovery period, a significant increase in body weight was noted in males at 1000 mg/kg bw/day. This was caused by a tendency of the control group animals to lose weight. One male in control group showed a significant decrease in body weight during the recovery period. However, no other abnormalities were observed in this male.

FOOD CONSUMPTION
No change was observed during the administration period in the test groups. A significant increase in food consumption was found in satellite females of the 1000 mg/kg bw/day group on Day 14 of administration. However, it was regarded as an incidental finding since the food consumption of the corresponding control group was relatively small on that day. Therefore, this change was not regarded as a compound-related effect.

HAEMATOLOGY
No changes were observed. After administration, a significant decrease in APTT was observed in males of the 100 and 300 mg/kg bw/day groups in comparison with control group. However, this change was not regarded as compound-related, because no dose-dependency was observed and this was within reference range.

CLINICAL CHEMISTRY
A significant decrease in inorganic P was observed in females of the 300 and 1000 mg/kg bw/day groups after administration. However, this change was not regarded as compound-related, since a dose-dependency was not observed and almost all the individual data were within the reference ranges except for one female of 300 mg/kg bw/day.
Choline esterase was increased in females of 1000 mg/kg bw/day after the recovery period but this was within the reference range. This change was not compound-related effect since this change was slightly and no change was observed after the administration period.

URINALYSIS
No significant changes were found during the administration and recovery periods.

NEUROBEHAVIOUR
No abnormality was observed for the sensory/reflex function, landing foot, grip strength and motor activity during the administration period. During the recovery period, motor activity increased from 0 to 60 min but not from 0 to 30 min in females dosed 1000 mg/kg bw/day. This effect was not compound-related since the observed change was slight and no change was observed during and after the administration period.

ORGAN WEIGHTS
After the administration period, a significant decrease in the absolute weight of seminal vesicles in males given and a significant decrease in relative weight of spleen in females were observed in the 100 mg/kg bw/day group. However, this change was not compound-related because no abnormalities were found at histopathological examination and there was no dose-dependency.
After the recovery period, a significant decrease in relative weight of pituitary in males of the 1000 mg/kg bw/day group and relative weight of thyroid in females of the 1000 mg/kg group bw/day was observed. Nevertheless, this effect was not compound-related because no abnormality was reported regarding these organs after administration period.

GROSS PATHOLOGY
No test substance-related changes were found. No abnormalities were found in breeding pairs which were not successful at mating and in those which were successful at mating but not pregnant.
After administration period, reddish thymus was noted in one male of the control group and subcutis mass was found in one female of 300 mg/kg by/day group (see also HISTOPATHOLOGY: NEOPLASTIC). After the recovery period, larger spleen and capsular thickening in spleen was found in one male and reddish area in thymus was observed in one female at 1000 mg/kg bw/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
No test substance-related changes were observed. There were also no changes regarding spermatogenic cycle.
Myocardial degeneration/fibrosis, foam cell accumulation in lung, mineralization in artery in lung, fatty degeneration of hepatocyte, microgranuloma in liver, solitary cyst in kidney, hyaline cast in kidney, lymphocyte infiltration in cortex of kidney, mineralization of cortico-medullary junction in kidney, fibrosis of cortex in kidney and haemorrhage in thymus were observed both in the control and 1000 mg/kg bw/day groups or only in the control group with low incidence. Hyaline droplet of proximal tubular epithelium in kidney was found in all males of the control and 1000 mg/kg bw/day groups. Brown deposit pigment and extramedullary haematopoiesis in spleen were found in all males and females of the control and 1000 mg/kg bw/day groups. However, there were no differences between control and test group. In the 1000 mg/kg bw/group, lymphocyte interstitium infiltration in prostate was found in one male, interstitial focal inflammation in lung and focal necrosis in liver was observed in one female. These changes occur naturally and were not compound-related.
No abnormalities were found in uterus and ovary in the non-pregnant females and the unsuccessful copulation females of the control and 1000 mg/kg bw/day groups, respectively. Degeneration of seminiferous tubules of testis, decrease in sperm and atrophy in prostate was observed in one control male.

HISTOPATHOLOGY: NEOPLASTIC
Mass on abdomen was found in a female after 40 days of administration in the 300 mg/kg bw/day group. However, this subcutaneous tumour of the mammary gland was a benign fibroadenoma and generated naturally.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion