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Description of key information

The NOAEL for systemic toxicity was found to be 500 mg/kg/day in rats of both sexes upon oral administration of the test item.

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:
28.03.2017-21.07.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 29, 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Specific Pathogen Free
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Inc., 322, Galmachi-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do 13201, Republic of Korea
- Age at the start of administrationn: 10 wks (males), 12 wks (females)
- Weight at the start of administration: Males: 353.4–406.6 g, Females: 216.7–288.1 g
- Fasting period before study: not specified
- Housing: Stainless wire mesh cages, 260W×350D×210H (mm); Polycarbonate cage, 260W×420D×180H (mm)
- Number of animals per cage: two animals (during quarantine-acclimation period); one animal (during dosing period)
- Diet (e.g. ad libitum): ad libitum; pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C)
- Water (e.g. ad libitum): ad libitum; public tap water, filtered and irradiated by ultraviolet light
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1–24.4°C
- Humidity (%): 39.0–60.7%
- Air changes (per hr): 10–15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
Route of administration:
oral: gavage
Details on route of administration:
Animals were randomly assigned to groups in an attempt to equalize mean group body weights.
A temporary identification number was marked on the tail using a red indelible pen.

Individual doses were calculated for each animal based on the most recently
recorded body weight data at a dose volume of 2 mL/kg. Dosing solutions were
administered once daily using 1 or 3 mL disposable syringes fitted with a gastric
intubation tube.
Control animals were dosed with vehicle only (corn oil).
Vehicle:
corn oil
Details on oral exposure:
Males of the main group were dosed once daily for a total of 49 days (for 2 weeks
prior to mating, during 2 weeks of mating and 21 days of post-mating).
Also, males and females of the recovery groups were dosed once daily for 49 days.
Females of the main group were dosed once daily for 2 weeks prior to mating until
Postpartum Day 13. Also, females showing no evidence of parturition signs were
dosed until Gestation Day 25.
The control group animals were dosed with the vehicle only with the same dose
volume as the test substance-treated groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the treatment period, formulation samples were prepared and analysed for stability and
homogeneity of the test item in the selected vehicle.
Verification of dose level concentrations were conducted using Gas Chromatography
Duration of treatment / exposure:
Males of the main group were dosed once daily for a total of 49 days (for 2 weeks prior to mating, during 2 weeks of mating and 21 days of post-mating).
Also, males and females of the recovery groups were dosed once daily for 49 days.
Females of the main group were dosed once daily for 2 weeks prior to mating until Postpartum Day 13.
Also, females showing no evidence of parturition signs were dosed until Gestation Day 25.
Frequency of treatment:
7 days per week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control group
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
mid dose group
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
high dose group
No. of animals per sex per dose:
- 12 animals per sex per group
- additional 6 animals per sex per group for the recovery groups (control and high dose)
Control animals:
yes, concurrent vehicle
Details on study design:
120 animals (60 males and 60 females) were included in the study (at the start of administration).
All animals were examined for general health upon receipt.
Body weights were recorded on the days of receipt, the last day of the quarantine-acclimation period and the day of group assignment.
All animals were quarantined and acclimated for 7 days and observed once daily for general health condition and clinical signs.
After the quarantine-acclimation period, all animals were observed once daily for clinical signs until group assignment.
Following group assignment, animals were uniquely identified by a blue indelible marking on the tail. A color-coded cage card was placed on each animal’s cage
displaying the group and dose level information.
Animals were randomly assigned to groups in an attempt to equalize mean group body weights.

- Dose selection rationale:
According to the results of a previous dose range finding study the doses were selected for the 3 dose groups.

- Body weights:
-- Body weights of males of the main group and animals of both sexes of the recovery groups were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing and recovery periods, the day prior to necropsy and on the day of necropsy (fasted body weights).
-- Body weights of females of the main group were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period, the day prior to necropsy and on the day of necropsy.
Fasted body weights recorded on the day of necropsy were presented, but were not included in the statistical analysis.

- Food consumption:
-- Food consumption of males of the main group and animals of both sexes of the recovery group was recorded just prior to dosing on Day 0 (one day before first
dosing), once a week during the dosing and recovery periods (except during mating) and the day prior to necropsy.
-- Food consumption of females of the main group was recorded just prior to dosing on Day 0 (one day before first dosing), once a week throughout the dosing period and the day prior to necropsy. Residual feed was recorded on the next day.
Individual food consumption was calculated by subtracting the amount of residual feed from the amount presented.

Positive control:
not applicable
Observations and examinations performed and frequency:
- Clinical signs: All animals were observed for general condition and clinical signs at least once
daily throughout the study. All animals were observed for moribundity and mortality twice daily.

- Observation of detailed clinical signs: All animals were observed prior to dosing and once weekly for the dosing and
recovery periods.
The following items were observed:
-- Skin, fur, eyes, mucous membranes, occurrence of secretion and excretion
-- Sutonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern, etc.)
-- Canges in gait, posture and response to handling, and the presence of clonic or tonic movements
-- Stereotypes (excessive grooming, repetitive circling, etc.) or bizarre behavior (self-mutilation, walking backward, etc.)

- Sensory function and motor activity:
Six males and six females were randomly selected from the main groups in addition to all recovery animals and evaluated a few days before necropsy for
pinna reflex, auditory (sound) reflex, corneal reflex, pupillary reflex, grip strength test and motor activity.

- Urinalysis:
Six males per group were randomly selected from the main group animals in addition to all male recovery animals for urinalysis two days before necropsy.
Fresh (3-hour) urine and 24-hour urine samples were collected from selected animals and analyzed.
Animals were fasted during the fresh urine collection, but were allowed free access to drinking water.
The following parameters were measured: pH, protein, glucose, bilirubin, occult blood, color and turbidity, sediment, urine volume mL, specific gravity.

- Hematology:
Blood samples were taken from all males and all females from the main groups and all animals from the recovery groups.
All animals were fasted for approximately 18 hours prior to necropsy. At necropsy, all animals were anesthetized with isoflurane
and blood samples were collected from the abdominal aorta.
The following haematological parameters were examined:
Erythrocyte count (RBC), Hemoglobin (HGB), Flow cytometry, Cyanmethemoglobin Hematocrit (HCT), Mean corpuscular volume (MCV),
Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets (PLT), 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:
Blood samples were collected from the abdominal aorta.
The following parameters were analyzed for all males and all females from the main groups and all animals from the recovery groups:
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Blood urea nitrogen (BUN), Urease-GLDH,
Creatinine (Crea), Total bilirubin (T-Bili), Vanadate oxidation, Total protein (TP), Albumin (Alb), Globulin (Glo), A/G ratio, Total cholesterol (T-Chol), Triglycerides (TG), Glucose (Glu), Calcium (Ca), Potassium (K), Ion-Sodium (Na), Chloride (Cl).

- Thyroid hormone analysis:
Blood samples were taken based on the following schedule:
- from all adult males and dams at termination
Animals were fasted for more than 18 hours before necropsy and anesthetized with isoflurane. Blood samples were collected from the abdominal aorta.
Thyroid hormones (T4 and TSH) were preferably measured as “total”.
The following parameters were analyzed: Total thyroxine (T4), Thyroid stimulating hormone (TSH).



Sacrifice and pathology:
- Necropsy:
-- The males of the main group were sacrificed on Day 50, and females of the main group were sacrificed on Postpartum Day 14.
-- All animals of the recovery groups were sacrificed two weeks after the final dosing.
-- All surviving animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anesthesia.
-- Complete gross postmortem examinations were conducted on all animals including the external surface and internal organs. All grossly visible
abnormalities were recorded.

- Organ weights:
-- 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.
The following organs were weighed from all males and females from the main groups and recovery groups: Brain, Heart, Liver, Thymus, Spleen, Thyroid,
Adrenal, Kidney, Uterus, Ovary, Testis, Epididymis, Prostate + seminal vesicle with coagulating gland.

- Tissue preservation and slide preparation:
- All males and all females from the main groups in addition to all animals of the recovery groups were selected for tissue preservation.

- Histopathology:
Histopathological examinations were conducted as follows:
-- At least six males and six females from the control, low, mid and high groups (especially focused on spermatogenesis and interstitial testicular cell structure)
-- All tissues from animals found dead or killed in a moribund condition during the study.
-- All gross, macroscopic lesions
Statistics:
The statistical analysis of this study was conducted using the SAS program.
For the data including body weights, food consumption, thyroid hormone value, urine volume, hematology and
clinical chemistry parameters, organ weights, grip strength, motor activity, the Bartlett test was conducted to analyze for homogeneity of variance
(significance level: 0.05).
One-way analysis of variance (ANOVA) test was applied on homogeneous data, if significant (significance level: 0.05),
the Dunnett’s t-test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
The Kruskal-Wallis test was applied on heterogeneous data, then if significant (significance level: 0.05),
the Steel’s test was performed for multiple comparisons (significance levels: 0.05 and 0.01, two tailed).
The data of sensory function were analyzed utilizing the Fisher’s exact test (significance levels: 0.05 and 0.01).
For the data of recovery group, the Folded-F test was applied to analyze homogeneity of variance (significance level: 0.05, two-tailed).
The Student t-test was applied for homogeneous data, but if overruled, the Aspin-Welch t-test was applied (significance
levels: 0.05 and 0.01, two-tailed).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In the main groups, salivation was observed in five males and three females at 500 mg/kg/day.
In the recovery groups, salivation was temporarily observed in one male and one female at 500 mg/kg/day.
However, salivation was considered to have little toxicological significance since it was
caused by physicochemical characteristics.
In the main group, hematuria was observed in one female at 500 mg/kg/day from Post-partum Day (PPD) 6 to the end of dosing.
It was considered to be incidental.

No clinical signs were observed in males and females of the main groups and the recovery groups in the detailed examinations as carried out once a week.
Mortality:
no mortality observed
Description (incidence):
All animals of the main and the recovery groups survived the duration of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in body weight changes were noted in males and females of
the main and the recovery groups when compared to the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the main groups, statistically significant decreases in food consumption were temporarily noted in males at 50 and 150 mg/kg/day on Day 36
and/or Day 43.
A statistically significant increase in food consumption was temporarily noted in females at 500 mg/kg/day on Gestation Day (GD) 14.

However, these statistical significances in food consumption decreases had little toxicological relevance since they were not related to any body weight changes.
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):
No effects were observed in any animal in the main and the recovery groups.
Other statistical significances were considered not to be test substance-related changes
because of a small difference and/or the values were within the range of historical
reference data.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No adverse effects were observed in any animal in the main and the recovery groups.
Other statistical significances were considered not to be test substance-related changes
because of a small difference and/or the values were within the range of historical
reference data.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In the main groups, occult blood and erythrocytes were observed in the urine in two males at 500 mg/kg/day.
These two males also showed an increase in the amount of urine (about 5-15 % vs. 500 mg/kg/day group).
Two male animals at 500 mg/kg/day in the recovery group were noted with occult blood and showed an increase in the amount of urine (about 26-
28 % vs. 500 mg/kg/day group).

These findings were considered transient but test substance-related as these findings
were not found in any animal of the mid or low dose group.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No test substance-related effects on auditory reflex, pinna reflex, pupillary reflex and
corneal reflex test were observed in animals of both sexes in the main and the recovery
groups when compared to the control group.

In the main and the recovery groups, there were no test substance-related effects in the
grip strength test when compared to the control group.

In the main and the recovery groups, there was some statistical significance in the
spontaneous motor activity. However, the statistical significance had little toxicological
relevance because it was of small difference or temporary change and showed no dose dependency.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related adverse effects on organ weights were observed in any animal
in the main and the recovery groups.
Other statistical significances in the absolute and/or relative organ weights were
considered not to be test substance-related effects because of a small difference and/or
the values were within the range of historical reference data.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One female at 50 mg/kg/day showed a stress-related gross finding such as a small thymus.
It was considered not to be related to the test substance since a dose-response relationship was not observed.

The other macroscopic findings in the adult animals were considered to be incidental
and not related to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Test substance-related changes were not observed in this study.

A moderate cortical atrophy of the thymus was noted in one female at 50 mg/kg/day. It was considered not to be
related to the test substance since a dose-dependent response was not clear, and this non-specific finding was frequently noted
in animals of poor condition under stress.

In addition, occult blood and erythrocytes in urine were detected in two males at 500 mg/kg/day, and they were correlated with pyelitis and chronic
progressive nephropathy, respectively.
This finding was considered incidental and of no toxicological significance since there were no common microscopical findings related to
test substance at 500 mg/kg/day.

All microscopic findings seen in other organs and tissues were considered to be incidental and of no toxicological significance.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis:
There were no test substance-related adverse effects in total thyroxine (T4) and TSH
levels in adult males of the main and the recovery groups.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Key result
Critical effects observed:
no
Conclusions:
The NOAEL of Frambinon Methyl Ether for systemic toxicity was considered to be 500 mg/kg/day for the animals of both sexes.
Executive summary:

The aim of this study performed according to OECD TG 422 and in compliance to GLP, was to assess the possible adverse effects of the substance on male and females Sprague-Dawley rats after repeated dose administration by oral gavage with dose levels of 50, 150, and 500 mg/kg body weight/day.

The animals of both sexes in the main and the recovery groups all survived the duration of the study.

No test substance-related adverse effects were noted in the results of body weights, food consumption, sensory function, motor activity, urinalysis, hematology, clinical chemistry, organ weights and thyroid hormone analysis in adult animals of both sexes in the test substance-dosed groups.

Salivation was observed in males and females at 500 mg/kg/day in the main and the recovery groups during the dosing period, but it was not considered to have toxicological significance since it was caused by physiochemical characteristics. Small thymus (cortical atrophy) was observed in one female at 50 mg/kg/day, it was considered not to be a test substance-related effect since there was no dose-dependent relationship. Occult blood and erythrocytes in urine were detected in two males at 500 mg/kg/day of the main group, and they were correlated with pyelitis and chronic progressive nephropathy, respectively. This finding was considered incidental and of no toxicological significance since there were no common microscopical findings related to test substance at 500 mg/kg/day. Still, two more male animals at 500 mg/kg/day in the recovery group were noted with occult blood.

These four males showed an increase in the amount of urine (about 5 % up to 28 % vs. 500 mg/kg/day group). Thus, these findings were considered transient but test item-related as these findings were not found in any animal of the mid or low dose group.

In conclusion, the NOAEL of Frambinon Methyl Ether for systemic toxicity was considered to be 500 mg/kg/day for the animals of both sexes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
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

For this endpoint there is one study available with data on toxicity after repeated dosing. The key study is a recent OECD 422, in which the substance was tested in a repeated dose assay via oral gavage to Sprague-Dawley rats with dose levels of 0, 50, 150 and 500 mg/kg bw/day.

No test substance-related adverse effects were noted in the results of body weights, food consumption, sensory function, motor activity, urinalysis, hematology, clinical chemistry, organ weights and thyroid hormone analysis in adult animals of both sexes in the test substance-dosed groups.

Findings of salivation in the highest dose group were not considered of toxicological significance as they were caused by physiochemical characteristics. One animal in the low dose group was found to have thymus cortical atrophy. This was considered non-test substance related since there was no dose-dependent relationship. Findings of occult blood and erythrocites in the urine, some related with pyelitis or chronic progressive nephropathy of animals in the highest dose group were considered incidental and of no toxicological significance since there were no common microscopic findings. The same animals showed an increase in urine volume. This was considered transient but test item-related.

In conclusion, the NOAEL for systemic toxicity was found to be 500 mg/kg/day for the animals of both sexes.

Justification for classification or non-classification

In section 3.9.2 of the EU regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP), the criteria are depicted for the classification of a substance for repeated exposure.

Based on the available information there was no specific target organ toxicity after 28 day of dosing. The NOAEL for repeated dose toxicity is considered to be 500 mg/kg bw/day. Therefore the substance is not considered to be classified.