Registration Dossier

Administrative data

Description of key information

Oral (OECD 407), rat, males ≥ 108.6 mg/kg bw/day (0.1% in diet)

Oral (OECD 407), rat, females ≥ 96.0 mg/kg bw/day (0.1% in diet)

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
Remarks:
with special focus on reproductive organs and tissues
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Feb - 29 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted: 3 Oct 2008
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
(Crl:CD(SD)), SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 191.8 - 209.5 g; females: 137.1 - 165.0 g
- Housing: individually in stainless steel wire mesh cages (260W×350D×210H mm)
- Diet: LabDiet® CERTIFIED RODENT DIET 5002, powder type; ad libitum
- Water: public tap water filtered and irradiated by ultraviolet light, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 - 24.4
- Humidity (%): 44.0 -58.8
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light):12/12

Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
DIET PREPARATION
The required amount of the test substance was weighed and mixed with required amount of corn oil using a vortex mixer. The required amount of powder feed, was mixed with the test substance formulation using a ball mill.
- Storage temperature of food: 4.3 - 6.3 °C

VEHICLE
- Concentration in vehicle: 10 mL of vehicle / 1 kg of powder feed
- Lot/batch no.: MKBV2080V, MKBZ9899V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted using a Gas Chromatography (GC-2010series, Shimadzu Corp., Japan). Samples were taken three times from the middle of each dosing formulation and analyzed for verification of dose level concentration prior to dosing. As a result, the accuracies at 0.01, 0.03 and 0.1% were 112.60, 109.67 and 98.84%, respectively. These results were within the acceptable range (range: ±15% of nominal values).
Duration of treatment / exposure:
28 days
Frequency of treatment:
ad libitum, daily, 7 days a week
Dose / conc.:
0.01 other: %
Remarks:
corresponding to 11.5 and 10.7 mg/kg bw/day in males and females, respectively
Dose / conc.:
0.03 other: %
Remarks:
corresponding to 32.8 and 32.2 mg/kg bw/day in males and females, respectively
Dose / conc.:
0.1 other: %
Remarks:
corresponding to 108.6 and 96.0 mg/kg bw/day in males and females, respectively
No. of animals per sex per dose:
5 for main and recovery group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the results of a dose range finding study where a test substance-related decrease in body weight (about 20% in males and 10% in females), as well as a liver weight increase of about 15%, were observed in males and females in the 0.3% groups. Therefore, 0.1% was set as the high dose level. Mid and low dose levels were selected at 0.03 and 0.01%, respectively. The control animals received the standard basal powder feed with corn oil (vehicle).
- Post-exposure recovery period in satellite groups: 2 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs and twice daily for mortality/moribundity
- Cage side observations: clinical signs, mortality, moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed for detailed clinical signs prior to dosing and once weekly during the observation period; observation included: Skin, fur, eyes, mucous membrane, occurrence of secretion, excretion; autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern, etc.); changes in gait, posture and response to handling, and the presence of clonic or tonic movement; stereotypy (excessive grooming, repetitive circling, etc.) or bizarre behavior (selfmutilation, walking backward, etc.)

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded just prior to dosing on Day 1, once a week during the
dosing and recovery periods and on the day of necropsy. Body weight data recorded on the day of necropsy was not included in the evaluation of body weights since these data are body weights of fasting animals.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: yes

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 (main group) and Day 43 (recovery group)
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes; 18 h prior to necropsy
- How many animals: 5 per control and main groups
- Parameters checked: total erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), total leukocyte count (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), basophils (BASO), reticulocytes (Reti), prothrombin time (PT), activated partial thromboplastin time (APTT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 (main group) and Day 43 (recovery group)
- Animals fasted: Yes; 18 h prior to necropsy
- How many animals: 5 per control and main groups
- Parameters checked: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (Crea), total bilirubin (T-Bili), total protein (TP), albumin (Alb), globulin (Glo), A/G ratio, glucose (Glu), total cholesterol (T-Chol), triglyceride (TG), phosphorus (P), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), Triiodothyronine (T3), Total Thyroxine (T4)


URINALYSIS: Yes
- Time schedule for collection of urine: Fresh (3-hour) urine and 24-hour urine samples were collected from all animals of both sexes per group in the main group and from all animals in the recovery group at Week 4 and Week 6.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Animals were fasted during the fresh urine collection, but were allowed free access to drinking water.
- Parameters checked: in fresh urine samples: pH, protein, glucose, ketone body, bilirubin, occult blood, color and turbidity, sediment; in 24-hour urine samples: urine volume, specific gravity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4 (main group) and Week 6 (recovery group)
- Dose groups that were examined: all main and recovery groups
- Battery of functions tested: Auditory, visual and proprioceptive stimuli (visual response, proprioceptive stimulus, auditory and pain responses, aerial righting reflex and hindlimb landing foot splaygrip strength); grip strength (using push-pull gauge for forelimbs and hindlimbs); motor activity (using activity monitor)

IMMUNOLOGY: No


OTHER:
Observation of estrus cycle:
The observation of estrus cycle for females was conducted on all females of the main group from Week 3 to Week 4 and of the recovery group from Week 5 to Week 6. For the examination of estrous cycle, the vaginal smear was conducted in the morning for approximately 14 days. Prepared smear of the vaginal mucosa was stained with Diff-Quick stain. Stained vaginal mucosa smear was examined using a light microscopy.

Sperm examination:
- Examination of caudal epididymal sperms:
Sperm motility was evaluated at necropsy with all males. The epididymides were weighed. The left caudal epididymis was pierced and placed in DPBS (Dulbecco’s Phosphate Buffered Saline) contacting 1% BSA (Bovine Serum Albumin). Sperm was incubated for approximately 3 to 10 min in the 1% BSA-DPBS culture media (approximately 37°C). Samples were placed on glass slides and the following parameters were analyzed using a sperm analyzer (HTM-TOX IVS, Hamilton Thorne Biosciences, U.S.A.):
MOT (motility, %), VAP (velocity of average path, μm/s), VSL (velocity straight line, μm/s), VCL (velocity curvilinear, μm/s), ALH (amplitude of lateral head displacement, μm), LIN (linearity, LIN = VSL / VCL × 100), STR (straightness, STR = VSL / VAP × 100), BCF (beat-cross frequency, Hz), Elongation (%), Area (μm sq), Rapid (rapid sperm, %), Medium (medium sperm, %), Slow (slow sperm, %), Static (static sperm, %)
Sperm sample was smeared on the glass slide, stained with Diff-Quick and examined for morphology using a light microscope (approximately 200 sperms/smear). Malformation was calculated as follows: Sperm malformation (%) = (Number of abnormal sperm/Number of total sperm (200)) × 100

- Testicular spermatid head counts:
Total sperm counting was evaluated at necropsy with all males. The right testis was weighed and was placed in 10 mL of distilled water, homogenized for one min and sonicated for three min. One drop of the homogenization-resistant sperm head solution was placed onto the sperm number Makler counting chamber and total sperm counts for each animal were determined using a microscope. The number of sperm per 1g of the right testis was calculated.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anesthesia for the main group on Day 29 and for the recovery group on Day 43. Complete gross postmortem examinations were performed on all animals including the external surface and internal organs. All grossly visible abnormalities were recorded.

ORGAN WEIGHTS: Yes
Paired organs were weighed together. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ-to-body weight ratios were calculated. Following organs were weighed: brain, heart, liver, kidney, testis, prostate (including seminal vesicle and coagulating glands), ovary, thyroid and parathyroid, thymus, spleen, adrenals, uterus

HISTOPATHOLOGY: Yes
The following organs and tissues were harvested and preserved in 10% neutral buffered formalin except for the testes, eyes and optic nerve which were fixed in Davidson fixative, and then preserved in 10% neutral buffered formalin:
brain, pituitary, thyroid, parathyroid, lung incl. bonchi, thymus, heart, trachea, liver, spleen, kidneys, adrenal, salivary gland (submandibular, sublingual and parotid glands), stomach, esophagus, duodenum, jejunum, ileum, cecum, colon, rectum, testis, epididymis, prostate, seminal vesicle and coagulating gland, ovary, vagina, uterus incl. cervix, submandibular lymph node, mesenteric lymph node, bone marrow (femur and sternum), spinal cord, sciatic nerve, eye, urinary bladder, sternum incl. bone marrow, mammary gland (inguinal), skin (inguinal), skeletal muscle (tight)

Histopathological evaluations of the samples were performed as follows:
- Organs or tissues from all animals of the control and high dose groups
- Organs with macroscopic lesions in the low dose group
Statistics:
Statistical analysis was performed using SAS Program (version 9.3, SAS Institute Inc., U.S.A.). Main groups: the data of body weight, food consumption, functional observations (hindlimb landing foot splay, grip strength and motor activity), urine volume, hematology, clinical chemistry, estrus cycle, examination of sperm (except for sperm malformation) and organ weights were analyzed utilizing Bartlett’s test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) was employed on homogeneous data; then, if significant, Dunnett’s test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneous data; then, if significant, Steel test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Main group of sperm malformation: Kruskal-Wallis test was employed on heterogeneous data; then, if significant, Steel test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Recovery group: the data of body weight, food consumption, functional observations (hindlimb landing foot splay, grip strength and motor activity), urine volume, hematology, clinical chemistry, estrus cycle, examination of sperm and organ weights were analyzed utilizing Folded-F test for homogeneity of variance (significance level: 0.05). Student t-test was employed on homogeneous data (significance level: 0.05) or Aspin-Welch t-test was employed for heterogeneous data (significance level: 0.05) for verifying significance (significance levels: 0.05 and 0.01, two-tailed).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Crust formation in left neck was observed in one male in the 0.1% group from Day 20 to Day 22. However, it was not considered to be a test substance-related effect since it was observed in only one animal incidentally. No detailed clinical abnormalities were observed in any dose group.
Mortality:
no mortality observed
Description (incidence):
All animals survived the duration of the study. There were no effects on mortality.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the dosing period, there were no statistically significant differences in body weights in females in the 0.01 and 0.03% groups when compared to the control groups. There were statistically significant decreases in body weights of males in the 0.01% group at Week 4, of males in the 0.03% group at Weeks 3 and 4, and of males in the 0.1% group from Week 1 to Week 4 and of females in the 0.1% group at Week 4 when compared to the control groups. During the recovery period, there were statistically significantly decreases in body weights of males in the 0.1% group when compared to the control group.
There were no statistically significant differences in body weight gain in females in the 0.01, 0.03 and 0.1% groups when compared to the control group. There were statistically significant decreases in body weight gain in males in the 0.01% group at Weeks 3 and 4, in males in the 0.03% group at Week 3, and in males in the 0.1% group from Week 1 to Week 4 when compared to the control group.
The mean body weights of males in the 0.1% group at the end of dosing period (Week 4) and of recovery period (Week 6) were 15.0% and 10.8% lower than those of the control groups, respectively. The decrease in body weights was correlated with the reduced food consumption in males in the 0.1% group. At the end of recovery period, a recovery of changes in body weight and food consumption was observed. In the absence of test substance-related effects in the other parameters, the decreased body weights and food consumption in males in the 0.1% group was considered as test substance-related non adverse effects.
In addition, all differences in body weight changes in the 0.01 and 0.03% groups were considered not to have toxicological significance since the magnitude of changes was within 10% of control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no statistically significant difference in food consumption in males and females in the 0.01% groups and in females in the 0.03% group when compared to the control groups. There were statistically significant decreases in food consumption in males in the 0.03% group at Weeks 1, 3 and 4, and in males from Week 1 to Week 4 and in females at Weeks 1 and 4 in the 0.1% groups.
During the dosing period, there were no statistically significant differences in the relative food consumption in both sexes in the 0.01% groups and in females in the 0.03% group when compared to the control groups. There were statistically significant decreases in the relative food consumption in males in the 0.03% group at Weeks 0 and 1, and in males at Week 1 and in females at Week 4 in the 0.1% groups when compared to the control groups. During the recovery period, there were statistically significant increases in the relative food consumption in males in the 0.1% group at Week 5 (recovery Week 1) when compared to the control group. At the end of recovery period, food consumption changes were observed to showa tendency of recovery. The changes in food consumption were correlated with the decrease in the mean body weights and considered as test substance-related changes.

The mean total test substance intakes at 0.01, 0.03 and 0.1% were 11.5, 32.8 and 108.6 mg/kg/day for males, and 10.7, 32.2 and 96.0 mg/kg/day for females, respectively.
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):
Test substance-related changes were not observed in males and females in the 0.01, 0.03 and 0.1% groups.
All differences in hematology parameters were judged to be produced due to biological variation or were considered to be unrelated to dosing of the test substance based on their small magnitude or because those changes were not observed in the main groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Test substance-related changes were not observed in males and females in the 0.01, 0.03 and 0.1% groups.
All differences in clinical chemistry parameters were judged to be produced due to biological variation or were considered to be unrelated to dosing of test substance because of inconsistency in both sexes and related parameters, and due to small magnitude or because those changes were not observed in the main groups.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Test substance-related changes were not observed in males and females in the 0.01, 0.03 and 0.1% groups.
All differences in urinalysis were judged to be biological variations or considered to be unrelated changes with dosing of the test substance based on their small magnitude or because the individual data showed minor variations within the historical reference range.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Test substance-related changes were not observed in males and females in the 0.01, 0.03 and 0.1% groups.
All differences in hindlimb landing foot splay, grip strength, ambulatory counts and vertical counts were considered to be changes unrelated to dosing based on their small magnitude or because those changes were not observed in the main groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Test substance-related changes were not observed in males and females in the 0.01, 0.03 and 0.1% groups.
The other organ weight changes, which were statistically significant, were not considered to be toxicologically important because of small magnitude, lack of morphological alteration or because those changes were not observed in the main groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Test substance-related changes were not observed in males and females in the 0.01, 0.03 and 0.1 % groups. All macroscopic findings were considered to be incidental and not related to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Test substance-related changes were not observed in males and females in the 0.1% groups. All other microscopic findings in various organs and tissues were considered to be incidental, spontaneous, and of no toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Observations of estrus cycle:
Test substance-related changes were not observed in females in the 0.01, 0.03 and 0.1% groups. No statistically significant differences in estrus cycle were noted in females in the 0.01, 0.03 and 0.1% groups when compared to the control group.

Examination of Sperm:
Test substance-related changes were not observed in males in the 0.01, 0.03 and 0.1% groups. All differences in sperm parameters were judged to be produced due to biological variation or were considered to be unrelated to dosing of the test substance based on small magnitude.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 108.6 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed up to the highest dose tested.
Remarks on result:
other: >= 0.1% in diet
Key result
Dose descriptor:
NOAEL
Effect level:
>= 96 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed up to the highest dose tested.
Remarks on result:
other: >= 0.1% in diet
Critical effects observed:
no
Conclusions:
Based on the results of this study, the NOAEL for systemic toxicity was ≥ 1% in diet (equivalent to 108.6 and 96.0 mg/kg bw/day in males and females, respectively), since no systemic adverse effects were observed in male and female rats after treatment. Additionally, no adverse effects on reproductive organs, estrus cycle and sperm parameters were observed.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
96 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

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

The test substance was tested in a 28-days repeated dose oral toxicity study according to OECD Guideline 407 and in compliance with GLP (2017). Sprague Dawley rats were treated with the test substance in the diet at dose levels of 0.01, 0.03 and 0.1% (equivalent to 11.5, 32.8 and 108.6 mg/kg/day for males, and 10.7, 32.2 and 96.0 mg/kg/day for females, respectively) for 28 days. 10 animals per sex and dose were allocated to the control and high dose group, 5 animals were allocated to the mid and low dose group. The control group received the vehicle corn oil. 5 rats per sex of the control and high dose group were designated to the recovery group and followed a 2–week treatment free period. The doses were selected on the basis of a repeated oral dose range-finding toxicity study in which a test substance-related decrease in body weight was observed in males and females in the 0.3% group.

Evaluated parameters included clinical signs, detailed clinical sign observations, body weight, food consumption, functional observations, urinalysis, hematology, clinical chemistry, observation of estrus cycle, examination of sperm parameters, gross post mortem examination, organ weights and histopathological evaluations of selected tissues.

All animals survived the duration of the study. There were no effects on mortality. No test substance-related toxic effects were noted in the results of evaluated parameters. Necropsy revealed no test substance related findings in males and females in any dose group. No adverse effects were observed in reproductive organs and tissues, sperm parameters of males, estrus cycle of females and thyroid hormones.

Based on the results of this study, the systemic NOAEL was ≥ 0.1 % in diet (equivalent to 108.6 and 96.0 mg/kg bw/day in males and females, respectively).

Justification for classification or non-classification

The available data on oral repeated dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.