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Description of key information

Under the test conditions (OECD 407 and GLP), the NOEL of the test substance was estimated to be 40 mg/kg/day in rats.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
other: “28-day Repeated Dose Toxicity Study in Mammalians” in “Partial Amendments to ‘Test Methods for New Chemical Substances’” (Kanpogyo Notification No. 700, Yakuhatsu Notification No. 1039, 61 Kikyoku Notification No. 1014 issued on December 5, 1986.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino Breeding Center, 735, Shimokomazuki, Hino-cho, Gamoh-gun, Shiga Prefecture 529-1633)
- Age at study initiation: 5 weeks
- Weight at study initiation: 139.4-158.7 g (males) and 119.0-139.5 g (females)
- Housing: individually housed in stainless steel wire-mesh floor cages (165 W x 300 D x 150 H mm; Tokiwa Kagakukiki K.K.)
- Diet: pellet food (MF from Oriental Yeast Co., Ltd.), ad libitum
- Water: (chlorinated) tap water supplied by Hita City, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2
- Humidity (%): 55±10%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hours (light from 7:00 to 19:00)
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test article was exactly weighed and dissolved in olive oil with stirring at 6.4 w/v%. Solutions of lower concentrations (1.6, 0.4 and 0.08 w/v%) were prepared by diluting the 6.4 w/v% solution. These solutions were prepared once a week.
- Dose volume : 10mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Chemical stability: Mean concentrations of the test substance determined immediately after preparations were within the range of 100 ± 10% of the specified concentration in both cases of 10.0 and 0.05 w/v% solutions. In addition, concentrations of the test substance in dose solutions determined 4 and 7 days after preparation were within 100 ± 10% of the concentrations determined immediately after preparation.
- Physical stability: The relative standard deviation of concentrations of the test substance in dose solutions sampled from different sites was not more than 5% for both 10.0 and 0.05 w/v% dose solutions.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
640, 160, 40 and 8 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a preliminary 14-day repeated-dose study conducted at 50, 150 and 1,000 mg/kg, abnormalities were revealed by blood chemical examinations in the 50 mg/kg group, by organ weights, necropsy and histopathological examinations in the 50 and 250 mg/kg groups, and by observation of general signs and symptoms, body weight, and hematological examinations in all groups. Based on these findings, the dose selection was made for the current study.
- Post-exposure recovery period in satellite groups: A recovery group was included in the 640 mg/kg group and the vehicle group and the recovery period was 14 days.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
All animals were observed at least once a day for general signs and symptoms.

BODY WEIGHT:
All animals were weighed on Day -2 before administration (upon grouping), on Days 1, 3, 8, 12, 17, 21, 26, and 28 during the treatment period, and on Days 1, 5, 10 and 14 of recovery during the recovery period. Animals were also weighed immediately before necropsy in order to calculate organ weights relative to body weights.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food intakes were determined in all animals once before starting treatment, on Days 3, 8, 15, 22 and 18 during the treatment period and on Days 4, 8 and 14 of recovery during the recovery period

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
Blood was sampled from the abdominal aorta of all animals under ether anesthesia after overnight fast (16-20 hrs) at the end of the treatment period and recovery period, and blood and plasma samples obtained were subjected to analysis of the parameters listed below. Since changes that suggest anemia were obtained in the preliminary study, bone marrow smears were prepared from the left femur of 3 males and 3 females per group selected in order of animal number at the end of the treatment period and recovery period, and bone marrow cells were classified in the vehicle control group and 640 mg/kg group at the end of the treatment period. Sodium citrate was used as anticoagulant to determine prothrombin time and activated partial thromboplastin time (APTT), while EDTA-2K was used for all other parameters. The following parameters were measured: Red blood cell count (RBC), White blood cell count (WBC), Hemoglobin concentration (Hb), Hematocrit (Ht), Mean cell volume (MCV), Mean cell hemoglobin (MCH), Mean cell hemoglobin concentration (MCHC), Platelet count (Platelet), Reticulocyte count (Reticulo), Prothrombin time (Pt), Activated partial thromboplastin time (APTT), Differentiation of leukocytes (Band neutrophils (N – Band), Segmented neutrophils (N-Seg), Eosinophils (Eosino), Basophils (Baso), Lymphocytes (Lymph), Monocytes (Mono)) Bone Marrow cell classification (myelogram) (Myeblast (Mybl), Promyelocyte (PMy), Myelocyte/metamyelocyte (My/Mt), Band/segmented neutrophils (N-Band, N-Seg), Eosinophils (Eosino), Basophils (Baso), Lymphocytes (Lymph), Plasmacytes (Plas), Megakaryocytes (Mk), Reticular cells (Ret), Mast cells (Mast), Monocytes (Mono), Proerythroblasts/basophilic erythroblast (PEb, BEb), polychromatophilic/orthochromatic erythroblasts (PoEb, NEb), Granulocytes/erythroid (M/E ratio))

CLINICAL CHEMISTRY:
Sera were separated from blood samples obtained at the same time as for haematology and sera obtained were subjected to analysis with respect to the following parameters: GOT, GPT, Alkaline phosphatase (ALP), Cholinesterase (ChE), gamma-GTP, Total cholesterol (T-Cho), Triglycerides (TG), Glucose, Total protein (T-Protein), Albumin, Albumin/globulin ratio (A/G ratio), Blood urea nitrogen (BUN), Creatinine, Total bilirubin (T-Bil), Calcium (Ca), Inorganic phosphorus (IP), Sodium (Na), Potassium (K), Chloride (Cl)

URINALYSIS:
Urinalysis was conducted once (Day 28) during the treatment period and once (Day 14 of recovery) during the recovery period. Sixteen-hour urine samples collected in individual metabolic cages were examined with respect to volume and color, while the following parameters were determined: pH, protein, ketone bodies, bilirubin, occult blood, sugar, and urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY:
All animals were examined in great detail. The following organs from all animals were weighed (wet weights): liver, kidneys, testes, ovaries, brain, spleen and adrenals.

HISTOPATHOLOGY:
- The organs and tissues listed below were collected from all animals: Lung, Stomach, intestines (duodenum, rectum), liver, Heart, Kidneys, urinary bladder, Testes, epididymis, prostate, seminal vesicles (including coagulating glands), ovaries, uterus, vagina, Brain (cerebrum, cerebellum), Bone marrow (femur), spleen, Pituitary thyroids (including parathyroids), adrenal glands, Eyes, and Gross lesions
- Collected organs and tissues were fixed in 10% neutral buffered formalin solution. Testes and epididymides were fixed in Bouin’s fixative. Organs and tissues from the following groups were examined under an optical microscope after preparing paraffin-embedded sections and staining with hematoxylin and eosin: Vehicle control: Stomach, intestines (Duodenum, Jejunum, ileum, cecum, colon, rectum), liver, heart, kidneys, brain (males only), spleen and adrenals; Vehicle control (recovery): Glandular stomach (females only), liver and adrenals (males only); 40 mg/kg: Liver (males only), 160 mg/kg: Glandular stomach (females only), liver and adrenals (males only); 640 mg/kg: Stomach, intestines (Duodenum, Jejunum, ileum, cecum, colon, rectum), liver, heart, kidneys, brain (males only), spleen and adrenals; 640 mg/kg (recovery): Glandular stomach (females only), liver and adrenals (males only). In addition, the skin of one animal in the 8 mg/kg group with macroscopic lesions was examined.
Statistics:
Data related to body weight, food intakes, hematological examinations, blood chemical examinations, urine volume, and organ weights were subjected to the test for quality of variance by Bartlett’s method, and one-way layout variance analysis was undertaken if equal variance was observed at a 5% significance level. If significant differences were observed in variance analysis, Dunnett’s test was conducted between the vehicle group and each treatment group. Kruskal-Wallis’ test was conducted if equal variance was not observed, while non-parametric Dunnett’s test was conducted between the vehicle group and each treatment group if significant differences were observed.
Details on results:
CLINICAL SIGNS AND MORTALITY
- Males: In the 640 mg/kg group, decreases in spontaneous locomotion and white turbid urine were observed in 10/12 and 12/12 males, respectively, while salivation occurred in 3/12, 4/6, 3/6, 5/6 and 12/12 males in the vehicle, 8 mg/kg, 40 mg/kg, 160 mg/kg and 640 mg/kg groups, respectively. Salivation onset patterns: Vehicle group: Salivation occurred sporadically or continuously immediately after treatment from Day 5 through Day 28; 8 mg/kg group: Salivation occurred sporadically or immediately after treatment from Day 6 through Day 26.; 40 mg/kg group: Salivation occurred sporadically immediately after treatment from Day 5 through Day 28; 160 mg/kg group: Salivation occurred sporadically or continuously immediately after treatment from Day 3 through Day 28; 640 mg/kg group: Salivation occurred continuously immediately after treatment from Day 2 through Day 28.
- Females: Abnormalities observed in females included decreases in spontaneous locomotion (9/12) and white turbid urine (12/12) in the 640 mg/kg group, salivation (5/6) and white turbid urine (4/6) in the 160 mg/kg group, salivation (6/12) in the vehicle group, salivation (2/6) and scab formation (1/6) in the 8 mg/kg group, and salivation (5/6) in the 40 mg/kg group. In addition, females from the 640 mg/kg group showed salivation (12/12) and loss of digits (forelimbs; 1/12). Salivation onset patterns: Vehicle group: Salivation occurred sporadically immediately after treatment from Day 5 through Day 27; 8 mg/kg group: Salivation occurred sporadically immediately after treatment from Day 5 through Day 27; 40 mg/kg group: Salivation occurred sporadically immediately after treatment from Day 8 through Day 26; 160 mg/kg group: Salivation occurred sporadically immediately after treatment from Day 5 through Day 28; 640 mg/kg group: Salivation occurred continuously immediately after treatment from Day 2 through Day 28.
- Recovery period: Both males and females showed no abnormality.

BODY WEIGHT AND WEIGHT GAIN
No abnormalties observed

FOOD CONSUMPTION AND COMPOUND INTAKE
Both males and females showed no abnormality.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION AND COMPOUND INTAKE
Not examined

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
At the end of the treatment period:
- Males: Hemoglobin concentrations and hematocrit decreased in the 640 mg/kg group, but no abnormality was observed in bone marrow cell classification. - Females: In the 40 mg/kg group, leukocyte differentiation showed decreases in the ratio of segmented neutrophils and increases in the ratio of lymphocytes. No abnormality was observed in bone marrow cell classification.
At the end of the recovery period:
- Males: No abnormality was observed.
- Females: Increases in platelet counts were observed in the 640 mg/kg group.

CLINICAL CHEMISTRY
At the end of the treatment period:
- Males: In the 640 mg/kg group, GPT and the A/G ratio increased. In addition, blood glucose levels decreased in the 40 mg/kg group and Na decreased in the 40 mg/kg and greater dose groups.
- Females: In the 40 mg/kg group, alkaline phosphatase increased, while the A/G ratio showed a tendency to increase and creatinine and Cl decreased. In the 40 and 160 mg/kg groups, urea nitrogen decreased.
At the end of the recovery period:
- Males: Ca increased in the 640 mg/kg group.
- Females: Total cholesterol increased in the 640 mg/kg group.

URINALYSIS
White turbid urine was sporadically observed in females from the 160 mg/kg and greater dose groups and males from the 640 mg/kg group. However, urine samples taken at the end of the treatment period for urinalysis showed no abnormality such as turbidity and the results of urinalysis were normal. The white urinary turbidity was thought to be of little toxicological significance because necropsy revealed no macroscopic changes in the urinary system and kidneys had no organic changes. It was thought to be due to precipitation of metabolites of the test article or other related compounds as a result of condensation of urine on the urine trays during the treatment period.

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
At the end of the treatment period:
- Males: Absolute and relative weights of liver increased in the 160 mg/kg and higher dose groups, while absolute weight of brain decreased in the 640 mg/kg group. The decrease in absolute weight of brain, was minimal and was considered to be incidental because histopathological examinations revealed no abnormality.
- Females: Absolute and relative weight of liver and relative weight of kidneys increased in the 640 mg/kg group.
At the end of the recovery period
- Both males and females showed no abnormality.

GROSS PATHOLOGY
At the end of the treatment period:
- Males: Enlargement of liver was observed in 1/6 and 6/6 animals, respectively, in the 160 mg/kg group and 640 mg/kg groups.
- Females: In the 640 mg/kg group, 1/6 females showed blackish mucosal regions in the glandular stomach, while 6/6 showed enlargement of liver. In addition, the skin of 1/6 females in the 8 mg/kg group showed loss of hair, while forelimbs of 1/6 females from the 640 mg/kg group showed a loss of digits.
At the end of the recovery period
- Both males and females showed no abnormality.

HISTOPATHOLOGY
At the end of the treatment period:
- Males: In the 160 mg/kg group, liver showed centrilobular hepatocyte hypertrophy (+, 1/6). In the 640 mg/kg group, liver showed centrilobular hepatocyte hypertrophy (++, 6/6), while adrenals showed granulation tissue associated with mineralization (+, 1/6).
- Females: In the 640 mg/kg group, liver showed centrilobular hepatocyte hypertrophy (+, 5/6; ++, 1/6), while glandular stomach showed necrosis of fundic mucosa (+, 1/6). In the 8 mg/kg group, skin showed scab formation (+, 1/1)
- These changes in adrenals and glandular stomach were not considered to be related to the test article since the former was a circumscribed, inveterate lesion, while the latter is often observed spontaneously.
At the end of the recovery period
- Both males and females showed no abnormality.
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The liver showed increases in weight, enlargement (macroscopic examination), and centrilobular hypertrophy of hepatocytes (histological examination) in males from the 160 mg/kg or larger dose groups.
Critical effects observed:
not specified
Conclusions:
Under the test conditions OECD 407 and GLP, the NOEL of the test substance was estimated to be 40 mg/kg/day in rats.
Executive summary:

A 28-day repeated-dose oral toxicity study of the test substance followed by a 14-day recovery study was conducted in groups of six 5-week-old male and female Crj:CD(SD)IGS rats (in compliance with OECD 407 and GLP). Four dose levels were used with the highest dose being 640 mg/kg followed by 160, 40 and 8 mg/kg. A recovery group was included in the 640 mg/kg group and the vehicle group. No animal died during the study, and no evidence of toxic effects of the test article was noted in terms of body weight and feed intake during the study period or urinalysis conducted at the end of the study period. In the observation of general signs and symptoms, males and females from the 640 mg/kg showed decreases in spontaneous locomotion during the treatment period. Hematological examinations conducted at the end of the treatment period showed decreases in hemoglobin concentrations and hematocrit in males from the 640 mg/kg group, while blood chemical examinations conducted at the end of the treatment period revealed increases in the A/G ratio in males and females and increases in GPT in males in the 640 mg/kg group in addition to increases in alkaline phosphatase and decreases in creatinine and Cl in females from the same group. Changes in organ weight observed at the end of the treatment period included increases in absolute and relative weight of liver in males from the 160 mg/kg or greater dose groups and females from the 640 mg/kg group and increases in relative weight of kidneys in females from the 640 mg/kg group. Necropsy revealed liver enlargement in males from the 160 mg/kg or greater dose groups and females from the 640 mg/kg group, while histopathological examinations conducted at the end of the treatment period showed centrilobular hypertrophy of hepatocytes in the liver of males from the 160 mg/kg or greater dose groups and females from the 640 mg/kg group. The recovery study showed no changes that persisted during the recovery period and at the end of the recovery period, suggesting that all changes observed at the end of the treatment were recoverable. The NOEL of the test substance was estimated to be 40 mg/kg/day in rats under the conditions of the present study because liver showed increases in weight, enlargement (macroscopic examination), and centrilobular hypertrophy of hepatocytes (histological examination) in males from the 160 mg/kg or larger dose groups.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
40 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline study, klimisch 2

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A 28-day repeated-dose oral toxicity study (Chemicals Evalutation and Research Insititute, 2000) of the test substance followed by a 14-day recovery study was conducted in groups of six 5-week-old male and female Crj:CD(SD)IGS rats (in compliance with OECD 407 and GLP). Four dose levels were used with the highest dose being 640 mg/kg followed by 160, 40 and 8 mg/kg. A recovery group was included in the 640 mg/kg group and the vehicle group. No animal died during the study, and no evidence of toxic effects of the test article was noted in terms of body weight and feed intake during the study period or urinalysis conducted at the end of the study period. In the observation of general signs and symptoms, males and females from the 640 mg/kg showed decreases in spontaneous locomotion during the treatment period. Hematological examinations conducted at the end of the treatment period showed decreases in hemoglobin concentrations and hematocrit in males from the 640 mg/kg group, while blood chemical examinations conducted at the end of the treatment period revealed increases in the A/G ratio in males and females and increases in GPT in males in the 640 mg/kg group in addition to increases in alkaline phosphatase and decreases in creatinine and Cl in females from the same group. Changes in organ weight observed at the end of the treatment period included increases in absolute and relative weight of liver in males from the 160 mg/kg or greater dose groups and females from the 640 mg/kg group and increases in relative weight of kidneys in females from the 640 mg/kg group. Necropsy revealed liver enlargement in males from the 160 mg/kg or greater dose groups and females from the 640 mg/kg group, while histopathological examinations conducted at the end of the treatment period showed centrilobular hypertrophy of hepatocytes in the liver of males from the 160 mg/kg or greater dose groups and females from the 640 mg/kg group. The recovery study showed no changes that persisted during the recovery period and at the end of the recovery period, suggesting that all changes observed at the end of the treatment were recoverable. The NOEL of the test substance was estimated to be 40 mg/kg/day in rats under the conditions of the present study because liver showed increases in weight, enlargement (macroscopic examination), and centrilobular hypertrophy of hepatocytes (histological examination) in males from the 160 mg/kg or larger dose groups.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only 28-day guideline study available, available as unpublished report, no restrictions, fully adequate for assessment.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Based on the findings in the repeated dose toxicity study, the substance is not classified according to Directive 67/548/EEC and according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.