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Description of key information

In an OECD combined repeated dose and reproductive /developmental toxicity screening test, the NOEL values for repeated dose toxicity obtained were 50 mg/kg bw/d for males and 500 mg/kg bw/d for females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-02-16 to 1997-05-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: An OECD guideline study, submitted to authorities.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory Japan Inc.
- Age at study initiation: 8 weeks old
- Weight at study initiation: ranging from 305 to 327 g for males and 201 to 225 g for females
- Fasting period before study: no
- Housing: except a mating period and a period from day 17 of gestation to day 4 of nurture, the animals were reared individually in metal mesh cages
- Diet: a solid diet (radiation sterilization diet NMF: Oriental Yeast Co., Ltd.), ad libitum
- Water: municipal tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 19.5 to 25.5°C
- Humidity: 41 to 66 %
- Air changes: 10 to 15 air changes per hour
- Photoperiod: 12-hour light / 12-hour dark cycle
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
- The administration volume was set to 0.5 mL/100 g to body weight. The administration liquid volume per individual was calculated based on a body weight measurement on each day in case of males. In case of females, the administration liquid volume per individual was calculated based on each body weight measurement in the pre-mating and mating periods, and then on day 0, day 7, day 14, and day 21 of gestation and on day 0 of nurture in the lactation period.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The administration period was 49 days by males starting 14 days before mating to day before autopsy and 54 days by females starting 14 days before mating, through mating period (14 days at maximum), gestation period, until day 3 of nurture (day before autopsy).
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 50, 150, 500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 males and 12 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dosage levels were selected based on results of a range-finding study (2 weeks oral administartion of 100, 300 and 1000 mg/kg bw/d of the test substance to rats). A high dose in the main test was set to 500 mg/kg bw/d which was approximate to a geometric mean of 1000 mg/kg bw/d and 300 mg/kg bw/d and which was estimated to represent toxicity signs but no deaths or serious pains. Subsequently, by subtracting it by the common ratio of about 3, a medium dose was set to 150 mg/kg bw/d, and a low dose was set to 50 mg/kg bw/d.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 times per days. Before administration, immediately after administration, and 2 hours after administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 3 times per days. Before administration, immediately after administration, and 2 hours after administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was measured on the day of the start of administration (before administration and on day 1 of administration), day 4, day 8, day 11, day 15, day 22, day 29, day 36, day 43, day 49 of administration, and the day of autopsy (day 50 after the start of administration).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption remaining quantity was measured on each of the same days as the days of the body weight measurements except the mating period and the day of autopsy. The quantity of food consumed per day was calculated from the difference between the remaining quantity and the feed quantity on the previous day.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the time of sacrifice and autopsy on the following day of the end of the administration period
- Animals fasted: Yes
- Anaesthetic used for blood collection: Yes
- The following parameters were examined:
Red blood cell count (RBC), Hemoglobin content (Hb), Hematocrit value (Ht), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Reticulocyte, Platelet, White blood cell count (WBC), Differential leukocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: same time as blood collection for the hematological examination
- Animals fasted: Yes
- The following parameters were examined:
Total cholesterol (T. cho), Triglycerides (TG), Phospholipid (PL), Iron (Fe), Total iron binding capacity (TIBC), Unsaturated iron binding capacity (UIBC), Total bilirubin (T. bilirubin), Glucose, Urea nitrogen (BUN), Creatinine, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (P), Total protein (TP), A/G ratio, Protein fractionation, GOT, GPT, LDH, γ-GTP, Blood plasma cholinesterase, Red blood cell cholinesterase.

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was conducted on week 7 of administration (day 45 and day 46 of administration)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (food withheld when in metabolic cage, water provided)
- The following parameters were examined:
4 and 20-hour urine volume, electrolytes, pH, specific gravity

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On the following day (day 50 after the start of administration) of the final administration, all existing animals were weighed, were subjected to blood collection under ether anesthesia, and underwent autopsy. The brain, sciatic nerve, heart, lung including bronchus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, pituitary, thyroid including glandula parathyroidea, adrenal, thymus, spleen, mesenteric lymph node, cervical lymph node, kidney, urinary bladder, testis, epididymis, seminal vesicle, prostate, mammary gland, skin, bone marrow including sternum and femur, and gross lesion locations were taken. The testis and epididymis were fixed in Bourin, and the other organs and tissues were fixed in 10 % formalin prepared with phosphate buffer.
Absolute organ weights of the brain, heart, lung including bronchus, thymus, liver, kidney, pituitary, thyroid including glandula parathyroidea, adrenal, spleen, testis, epididymis, seminal vesicle, and prostate were measured, and relative organ weights were calculated from the final body weight.

HISTOPATHOLOGY: Yes
For all animals, the organs and tissues taken in the autopsy were paraffin-embedded. For all animals in the control group and the high-dose group, the gross lesion locations in the autopsy were sectioned, were stained with haematoxylin and eosin, and were examined microscopically.
Statistics:
- Multiple Test
Homogeneity of variance among groups was tested by the Bartlett’s method. As a result, in a case where variance was homogeneous, the one-way analysis of variance was carried out. In a case where a significant difference was present among the groups, the paired comparison test between the control group and each treated group was carried out using the Dunnett’s method. In a case where variance was not homogeneous, the Kruskal-Wallis rank test was carried out. If the result was significant, the Dunnett’s test was carried out for a mean rank difference between the control group and each treated group.
Body weight, food consumption, quantitative items of urinalysis, count of estrus, estrous cycle, number of living-together days, gestation length, hematological examination values, blood chemical examination values, organ weights, number of corpora lutea, number of implantation sites, number of live born pups, implantation index, sex ratio, delivery index, live birth index, and viability index.
- x2 Test: Copulation index, fertility index, and birth index.
- Mann-Whitney’s U Test, Fisher’s Exact Test: The Mann-Whitney’s U test was conducted for expression frequency and increase in severity of histopathological findings. The Fisher’s exact test was conducted for pancreas acinus basophilic focus and lung osseous metaplasia.
Details on results:
CLINICAL SIGNS AND MORTALITY
Male and female rats: no changes in clinical signs related to administration of the test article were observed.

BODY WEIGHT AND WEIGHT GAIN
Male and female rats: the body weights of the treated groups changed almost similarly to those of the control group through out the administration period

FOOD CONSUMPTION
Male and female rats: no notable abnormalities

OPHTHALMOSCOPIC EXAMINATION - not examined

HAEMATOLOGY and CLINICAL CHEMISTRY
Haematology (male rats): significant low values for hemoglobin content, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were observed in the 500 mg/kg bw/d group. Although a significant low value for the mean corpuscular hemoglobin in the 150 mg/kg bw/d group and significant low values for the mean corpuscular volume and mean corpuscular hemoglobin in 50 mg/kg bw/d group were observed, the other erythroid parameters showed almost similar values to those of the control group. The other measurements showed no abnormalities.
Clinical chemistry (male rats): a significant low value for the α2-globulin fractionation ratio in the 50 mg/kg bw/d group, a significant high value for the urea nitrogen and a significant low value for the α2-globulin fractionation ratio in the 150 mg/kg bw/d group, and further increase in changes in the 150 mg/kg bw/d group. In the 500 mg/kg bw/d group a significant high value of GOT activity, a significant high values of blood serum total iron binding capacity, blood serum unsaturated iron binding capacity, and β-globulin fractionation ratio were observed.
In addition, although significant high values for the A/G ratio and albumin fractionation ratio in the 150 mg/kg bw/d group were observed, such changes were not seen in the 500 mg/kg bw/d group.

Female rats: no substance related abnormalities in haematology and clinical chemistry parameters

URINALYSIS
Male rats: significant high values for the total daily excretion volumes of sodium, potassium, and chlorine were observed in the 500 mg/kg bw/d group. In the 150 mg/kg bw/d and and the 50 mg/kg bw/d dose groups, there were no obvious differences from the control group.

Female rats: no substance related abnormalities in urinalysis parameters

NEUROBEHAVIOUR - not examined

ORGAN WEIGHTS
Male rats: a tendency toward a high value for an absolute weight of the liver, a significant high value for a relative weight of the liver, and significant high values for absolute and relative weights of the kidney in the 500 mg/kg bw/d group. A significant high value for a relative weight of the kidney in the 150 mg/kg bw/d group was observed as well as in the 50 mg/kg bw/d group.

Female rats: no substance related abnormalities

HISTOPATHOLOGY:
Male and female rats: no substance related abnormalities

GROSS PATHOLOGY
No substance related abnormalities

Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: in females there were no effects of the test substance on clinical signs, body weights, food consumption or histopathology in any treatment group.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD guideline compliant study.

Additional information

Repeated dose oral toxicity

A combined repeated dose and reproductive/developmental toxicity screening test was performed according to OECD guideline 422, with the test substance in the following concentrations: 0 (olive oil – vehicle), 50, 150, and 500 mg/kg bw/d. The test substance was administered orally to male rats for 49 days and to females for 54 days.

In the 500 mg/kg bw/d treated group (males), abnormal results were detected in the hematological examination and in the blood chemical examination: low values of hemoglobin content, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin, and high values in blood serum total iron binding capacity and blood serum unsaturated iron binding capacity in correlation with the results that showed low value of α2-globulin fractionation ratio and a high value of β-globulin fractionation ratio. Additionally, high value of urea nitrogen was observed. Abnormal results were obtained in organ weights examination; high values for absolute and relative weight of kidney. No effects were observed in clinical signs, body weights, food consumption, urinalysis, autopsy, and in the histopathological examination.

In the 150 mg/kg bw/d treated group (males), a high value of urea nitrogen and a high value of kidney relative weight were observed. No effects were observed in clinical signs, body weights, food consumption, urinalysis, hematological examination, autopsy, and in the histopathological examination.

In the 50 mg/kg bw/d treated group (males), no effects were observed in clinical signs, body weights, food consumption, urinalysis, hematological examination, blood chemical examination, and in the pathological examination.

By female rats, in the 50, 150, and 500 mg/kg bw/d treated groups, no effects of the test substance were observed in any of the examined parameters.

Therefore, under the conditions of the study, the no observed effect levels for general toxicity are considered to be 50 mg/kg bw/d for males and 500 mg/kg bw/d for females.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The only reliable available study

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)
The available studies were considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is considered to be not classified under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.
 
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is considered to be not classified under Regulation (EC) No. 1272/2008, as amended for the sixth time in Regulation EC 605/2014.