Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to test guideline and GLP compliance.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: 7-weeks-old
- Weight at study initiation: 186-255g males, 151-212g females
- Fasting period before study: Ad libitum
- Housing: After randomization, individually housed in suspended stainless-steel, wire-mesh cages measuring 24.2x22.0x17.3 cm
- Diet (e.g. ad libitum): 12 h/day since 1 week prior to study initiation
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 - 24.9 ºC
- Humidity (%): 40.3 - 68.1 %
- Air changes (per hr): 10 AC/h
- Photoperiod (hrs dark / hrs light): 13h light/11h dark (light from 6:00 to 19:00)
Route of administration:
oral: feed
Vehicle:
other: PMI Certified Rodent Diet
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): PMI Certified Rodent Diet
- Storage temperature of food: -20ºC
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method used to assay the level of the test article in the diet involved extraction of the test material from the feed mixture using an acetonitrile solvent and high-performance liquid chromatography.

A sample of each formulation from the middle of each barch of formulated diet prepared on study Days 1, 8, 29, 57, and 85 was analyzed in duplicate for concentration of the test material.

Stability of tran-anethole in rodent diet at 0.015 abd 3% has been determined at the following condicitons. 1) room temperature (animal room conditions) for 12 and 14 hours; 2) refrigerated in sealed amber jars for 6 and 11 days; and 3) frozen (-15 to -25ºC) for 1 month.

Homogeneity of trans-anethole in rodent diest at 0.015 and 3% was determined in the method validation study
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
600 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
900 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose based on study CHV 2595-102 (28-day range-finding study in rats)
- Rationale for animal assignment (if not random): Rats historically have been used in safety evaluation studies and are recommended by appropiate regulatory agencies
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily at the beginning of the lights-on phase of the daily light cycle.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily, with at least 6 hours between each observation period. A through physical examination was conducted once prior to treatment and weekly thereafter at the bedinning of daily light cycle.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded at randomization, prior to treatment, twice weekly for 4 weeks, and weekly thereafter.

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during week 12

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes (CO2/O2 inhalation)
- Animals fasted: No
- Parameters: differential leukocyte count and cell morphology, erythrocyte count, hematocrit, hemoglobin, leukocyte count, mean cell hemoglobin, mean cell hemoglobin concentration, mean cell volume and platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- Parameters: alanine aminotransferase, albumin, albumin/globulin ratio, alkaline phosphatase, aspartate aminotransferase, calcium, chloride, creatinine, gamma glutamyltransferase, globulin, glucose, phosphorus, potassium, sodium, sorbitol dehydrogenase, total bilirubin, total protein and urea nitrogen

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Examination of: all orifices, carcass, cranial cavity, external surface of the brain, cervical tissues and organs, thoracic-abdominal-pelvic cavities and their viscera, external body surface, nasal cavity and paranasal sinuses, external and cut surfaces of the spinal cord (at tissue trimming) and cut surfaces of the brain (at tissue trimming)

HISTOPATHOLOGY: Yes
Examination of: adrenals, aorta (thoracic), bone (two ribs and one femur), bone marrow (sternum), brain with brainstem (medulla/pons, cerebellear cortex, and cerbral cortex), colon, cecum, rectum, duodenum, jejunum, ileum, esophagus, eyes (with optic nerve and Harder's glands), heart, kidneys, larynx, lesions, liver, lung, mammary gland (inguinal), mandibular lymph node, meseneric lymph node, ovaries, pancreas, pituitary, prostate, salivary glands (mandibular), sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin, spinal cord (cervical, lumbar, and mid-thoracic), spleen, stomach, testes with epididymides, thymus, thyroid (parathyroids), tongue, trachea, urinary bladder, uterus and vagina
Other examinations:
Organ weights: adrenals, brain with brainstem, heart, kidneys, liver, lung, ovaries, pituitary (weighed postfixation), prostate, spleen, testes with epididymides, thymus, thyroid/parathyroid (weighed postfixation) and uterus

Tissue preservation: adrenals, aorta (thoracic), bone (two ribs and one femur), bone marrow (sternum), brain with brainstem (medulla/pons, cerebellear cortex, and cerbral cortex), colon, cecum, rectum, duodenum, jejunum, ileum, esophagus, eyes (with optic nerve and Harder's glands), heart, kidneys, larynx, lesions, liver, lung, mammary gland (inguinal), mandibular lymph node, meseneric lymph node, ovaries, pancreas, pituitary, prostate, salivary glands (mandibular), sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin, spinal cord (cervical, lumbar, and mid-thoracic), spleen, stomach, testes with epididymides, thymus, thyroid (parathyroids), tongue, trachea, urinary bladder, uterus and vagina
Statistics:
Weekly body weights, body weight change, weekly food consumption, total food consumption, efficiency of food utilization, clinical pathology data (except cellular morphology gradings), terminal body weights, and organ weight data of the trated groups were compared statistically to the date from the same sex of the control group.

If variances of untransformed data were heterogeneous, a rank transformation of the data was performed to achieve variance homogeneity . If the transformation did not achieve variance homogeneity, the analyses were still performed on the rank-transformed data.

Statistical significance (p =< 0.05) is designated throughout the text of this report by the term significant.
Clinical signs:
no effects observed
Description (incidence and severity):
The findings observed occurred sporadically and/or were of the type commonly seen in this species at the laboratory. There were no compount-related adverse effects on survival. All animals survived to scheduled sacrifice.
Mortality:
no mortality observed
Description (incidence):
The findings observed occurred sporadically and/or were of the type commonly seen in this species at the laboratory. There were no compount-related adverse effects on survival. All animals survived to scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower values comparing with the ones of control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower food consumption than control group.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Lower values comparing with the ones of control group.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The findings seen included iritis, chromodacryorrhea, and retinopathy. There is no indication of dose or compound-related ocular disease.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Decrease in the mean cell volume an mean cell hemoglobin for Group 5 females and for platelets for Group 5 males and females compared to the control values.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Significant treatment-related changes: higher mean values for gamma glutamyltransferase, alanine aminotransferase and aspartate aminotransferase and lower mean values for total protein, albumin and glucose.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increase in liver-to-body-weight percentages.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlarged liver in the males and various uterine findings in the females.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the findings which are directly related to treatment, excluding those resulting from diminishing palatability of the diet at higher dose.
Critical effects observed:
not specified
Conclusions:
Based on the findings which are directly related to treatment, excluding those resulting from the diminishing palatability of the diet at higher doses, the no-observable-adverse-effect level (NOAEL) is 300 mg/kg/day for females and male rates (based on an elevation in serum gamma glutamyltransferase at =>600 mg/kg/day in females, and an increased incidence of single cell necrosis at =>600 mg/kg/fay in males).
Executive summary:

This study was designed to evaluate the safety of trans-anethole when administered daily in the diet of rats for at least 90 days and to establish a NOAEL. Sprague-Dawley rats in Groups 2 -5 (20 rats/sex/group) received the test material in their food at target dose levels of 150, 300, 600, or 900 mg/kg/day, respectively. Rats in Group 1 (20 rats/sex) served as negative (vehicle) controls and received the basal diet only. Formulated diets were made available to the animals for approximately 12 hours daily (during the daily dark cycle). Assessment of potential toxicity was based on body weight gains, food consumption, efficiency of food utilization, ophthalmoscopic findings, clinical pathology findings (hematology, coagulation, and serum chemistry evaluations), gross pathology findings, and histopathological assessments.

Results of routine concentration analyses indicated that all formulations were within 5% of tarfet. Once the target dose levels were instituted, the avarage achieved compound consumptions for the Group 2 -5 males were 151.5, 303 .4, 602.9, and 892.7 mg/kg/day, respectively, whereas the achieved compound consumptions for the Group 2 -5 females were 151.5, 305.5, 605.8 and 888.1 mg/kg/day, respectively. In general, the dietary concentration of trans-anethole in each treatment group increased each week for the duration of the study. Greater difficulty was noted in achieving and maintaining the high target dose level (900 mg/kg/day) relative to the lower target dose levels, as a result of the adverse affect the test material had on diet palatability.

All animals survived to scheduled sacrifice. There were no obvious treatment-related clinical signs or ophthalmoscopic findings.

A number of findings, considered indirect treatment related findings, resulted largely from the reduced palatability of the diet, especially at the higher doses.

Several of the clinical pathology and histopathology findings, aslo considered indirect treatment-related findings, are associated with the treatment-related decrease in body weight and food consumption. In addition to these, certain clinical chemistry parameters and liver histopathology were directly associated with treatment.

Mild, although significant, increases in liver-to-body-weight ratops were noted in rats treated with trans-anethole at dose levels =>300 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat

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

Justification for classification or non-classification