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Diss Factsheets

Administrative data

Description of key information

Repeated Dose Toxicity: Oral

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non-toxic to living organisms when dosed repeatedly via oral route. The No Observed Adverse Effect Level can be considered to be >1000 mg/kgbw/day. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.

Repeated Dose toxicity: Inhalation

A short-term toxicity study need not be conducted as because exposure of humans via inhalation route in production and/or use is not likely based on the provided thorough and rigorous exposure.The estimated vapour pressure of test chemical at 25 deg C was observed to be 2.08E-17 mmHg (2.78E-15 Pa) Hence, this endpoint was considered for waiver.

Repeated Dose Toxicity: Dermal

A short-term toxicity study need not be conducted as because exposure of humans via dermal route in production and/or use is not likely based on the provided thorough and rigorous exposure assessment. The acute dermal toxicity value for the test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. The substance failed to cause any dermal irritation as well sensitization. Considering this, the end point for repeated dermal toxicity is considered as waiver.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Weight of evidence approach based on various test chemicals
Justification for type of information:
Weight of evidence approach based on various test chemicals
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: Weight of evidence approach based on various test chemicals
Principles of method if other than guideline:
Weight of evidence approach based on various test chemicals
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: 2. Sprague Dawley; 3. Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
other: 2.0.5% carboxymethylcellulose / sodium solution containing 0.1% Tween80; 3. corn oil
Details on oral exposure:
2. PREPARATION OF DOSING SOLUTIONS:The dosing solution was prepared by suspending the test substance in 0.5% carboxymethylcellulose / sodium solution containing 0.1% Tween80 and refrigerated. The test substance in the administration solution was stable for at least 8 days under refrigerated storage conditions, and it was confirmed that the used administration solution contained a uniform amount of the test substance.
DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% carboxymethylcellulose /
sodium solution containing 0.1% Tween80
- Concentration in vehicle: 0, 20, 140 or 1000 mg/kg/day
- Amount of vehicle (if gavage): The dosing volume was 10 ml / kg and was calculated based on the body weight of the closest measurement day. The solvent was similarly administered to the control group.
- Lot/batch no. (if required): No data available
- Purity: No data available
3. PREPARATION OF DOSING SOLUTIONS:The test item was weighed and dissolved in a vehicle(corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily.At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item. The details of dose formulation preparation is maintained in raw data.
DIET PREPARATION - Rate of preparation of diet (frequency): - Mixing appropriate amounts with
(Type of food):
- Storage temperature of food:
VEHICLE - Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0, 750, 1000 and 1250 mg/kg bw
- Amount of vehicle (if gavage): 2 mL/100g body weight
- Lot/batch no. (if required): A1708001 and A1703001
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
3. Specificity, Linearity, Precision (%RSD), Accuracy (% Recovery) and Homogeneity were analyze by using HPLC-UV.
Duration of treatment / exposure:
2. 28 days
3. 48 days (male rats, main study) Approx 63 days (female rats, main study) 66 days (recovery groups)
Frequency of treatment:
2. daily
3. daily
Remarks:
Study 2: 0, 20, 140, 1000 mg/kg/day
Remarks:
Study 3: G1, G1-R - 0, G2- 750, G3- 1000, G4,G4-R- 1250 mg/kg/day
No. of animals per sex per dose:
2. Males, 6 ; females, 6 per dose group
3. 13 rats per sex per dose (main study)
5 rats per sex per dose (recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
2. Dose selection rationale: As a dose-setting study, the test substance was orally administered at a dose of 0, 30, 100, 300, and 1000 mg / kg for 14 days in 4 males and 4 females per group. Observation of general condition, body weight, and food consumption , Hematology and blood biochemistry, autopsy, and organ weights were measured. As a result, no toxic effects due to administration were observed. Therefore, the maximum dose for this study was 1000 mg / kg / day, and the following three doses of 300 and 100 mg / kg / day and controls were set.
- Rationale for animal assignment (if not random): Prior to the start of treatment, animals were divided into groups by weight-based stratified random sampling.The number of animals in each group was 6 males and 6 males, and for the control and high-dose groups, a recovery group of 14 males and 6 males was established.
3. - Dose selection rationale: - Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing. The animals were allocated to the different test groups using the ‘Group Allocation’ function in the MS Excel Add-in “Daniel’s XL Toolbar” (http://xltoolbox.sourceforge.net/). Individual body weights were considered within ± 20% of the groups mean.
Positive control:
no data available
Observations and examinations performed and frequency:
2. CAGE SIDE OBSERVATIONS: Yes / No / Not specified : yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included. : All rats were observed daily for general condition.
DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified; yes
- Time schedule: daily
BODY WEIGHT: Yes / No / Not specified : yes
- Time schedule for examinations: Body weight was measured on the first day of administration and once a week thereafter
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified : yes, Food intake was measured on the first day of administration and once a week thereafter, and the average daily food intake per animal was calculated for each period
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 / No / Not specified ; not specified
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified : not specified
- Time schedule for examinations: not specified
OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified : no
HAEMATOLOGY: Yes / No / Not specified : yes
- Time schedule for collection of blood: At the time of each planned autopsy, blood was collected from the posterior vena cava under anesthesia by intraperitoneal administration of thiopental sodium
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified: thiopental sodium
- Animals fasted: Yes / No / Not specified : no data available
- How many animals: all the test and control animals
- Parameters checked in table [No.?] were examined.: red blood cell count (sheath flow DC impedance detection method), white blood cell count (RF/ DC impedance detection method), platelet count(Sheath flow DC impedance detection method), hemoglobin concentration (SLS hemoglobin method), hematocrit value (red blood cell pulse peak detection method) multi-item automatic blood cell analyzer, white blood cell percentage ( Wright stained smear) automatic blood cell analyzer, reticulocyte count (flow cytometry method using argon laser), automatic reticulocyte analyzer, Prothrombin time (PT: Quick one-step method), activated partial thromboplastin time (APTT: activated cephaloplastin method) blood coagulation automatic measuring device (KC 10A: Amerung) were measured.The mean red blood cell volume (MCV), average red blood cell pigment content (MCH), and average red blood cell pigment concentration (MCHC) were calculated from the results of the tests. As a coagulation inhibitor, 3.13% sodium citrate aqueous solution was used to measure prothrombin time and activated partial thromboplastin time, and EDTA-2K was used to measure other parameters
CLINICAL CHEMISTRY: Yes / No / Not specified : yes
- Time schedule for collection of blood: At the time of each planned autopsy, blood was collected from the posterior vena cava under anesthesia by intraperitoneal administration of thiopental sodium
- Animals fasted: Yes / No / Not specified : not specified
- How many animals: all the test and control animals
- Parameters checked in table [No.?] were examined.: The collected blood is allowed to stand at room temperature for about 30 minutes and then centrifuged at 3000 rpm for 10 minutes.Using the obtained serum, total protein (Biuret method), albumin (BCG method), A / G ratio (total protein and Calculated from albumin), glucose (GK-G6PDH method), triglyceride (LPL-GK-G3POPOD method), total cholesterol (CES-CO-POD method), urea nitrogen (Urease-GLDH method), creatinine (Jaff method) , Calcium (O-CPC method), inorganic phosphorus (UV method), GOT (SSCC improved method), GPT (SSCC improved method), γ-GTP (SSCC improved method), ALP (GSCC improved method), sodium, potassium, Crawl (ion selective electrode method) was measured with an automatic analyzer .
URINALYSIS: Yes / No / Not specified : yes
- Time schedule for collection of urine: fresh urine was collected from all surviving animals 2 days
before dissection at the end of administration
- Metabolism cages used for collection of urine: Yes / No / Not specified : no
- Animals fasted: Yes / No / Not specified: no
- Parameters checked in table [No.?] were examined. : pH, occult blood, protein, sugar, ketone bodies, bilirubin, urobilinogen (test paper method, Marti Sticks: Miles Sankyo Co., Ltd.) was measured. As a result, changes were observed in females in the 1000 mg / kg group, so the collected urine wasfurther collected for 21 hours only for females, the urine volume was measured with a graduated cylinder, and the specific gravity (refractive method) was measured with a urine hydrometer Atago Co., Ltd., sodium and potassium (flame photometric method) with a fully automatic flame photometer(FLAME-30C / AD-3: JASCO Medical Co., Ltd.) and chlor (coulometric titration method) with a
chloride meter (Model 925: Corning Medical Co., Ltd.) Similar examinations were performed on females with changes during the treatment period 2 days before dissection at the end of the recovery period.
3. CAGE SIDE OBSERVATIONS: Yes
- Time schedule:twice daily (morning and evening)
- Cage side observations checked in table [No.?] were included. morbidity and mortality, throughout the acclimatization and study period.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, preferably at the same time each day considering the peak period of anticipated effects after dosing.
BODY WEIGHT: Yes
- Time schedule for examinations:Males and females were weighed during randomization, on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), day 4 postpartum
and before terminal sacrifice.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as gfood/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations:
- Dose groups that were examined:
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Five males and five females, randomly selected from each group, just prior to necropsy.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, Animals were fasted overnight prior to blood collection.
- How many animals:Five males and five females, randomly selected from each group,
- Parameters checked in table [No.?] were examined.: Total Erythrocyte Count (RBC), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Hemoglobin (HGB), Mean Corpuscular Hemoglobin (MCH),Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (PLT), Total Leukocyte count(WBC), Prothombin Time (PT) and Activated Partial Thromboplastin time (aPTT) were examined. CLI
NICAL CHEMISTRY: Yes
- Time schedule for collection of blood:Five males and five females, randomly selected from each
group, just prior to necropsy
.- Animals fasted: Yes, Animals were fasted overnight prior to blood collection.
- How many animals:Five males and five females, randomly selected from each group,
- Parameters checked in table [No.?] were examined.: Glucose (Glu), Cholesterol (Chol), Triglycerides(TRIG), Alanine amino transferase (ALT), Aspartate amino transferase (AST), Calcium, Albumin (Alb), Total Protein (TP), Creatinine (Crea), Phosphorus, Urea, Sodium (Na), Potassium (K), Blood urea nitrogen (BUN), Globulin (Glob), Alb/ Glb (A:G), Bile acids, Thyroid hormones (Total T4 and TSH),Testosterone and Estrogen were examined. URINALYSIS: Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified- Parameters checked in table [No.?] were examined.Not specified
NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations:During the last week of treatment and that of recovery groups
- Dose groups that were examined: five males and five females from control and treatment groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity were assess.
IMMUNOLOGY: Not specified
- Time schedule for examinations:Not specified
- How many animals:Not specified
- Dose groups that were examined:Not specified- Parameters checked in table [No.?] were examined.: Not specified
OTHER:number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities
Sacrifice and pathology:
2. GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: At the time of each planned killing, all animals were bled and the abdominal aorta was severed, exsanguinated and necropsied, and the brain, liver, kidney, adrenal gland, thymus, spleen, testis and ovary were weighed. In addition to these organs, the pituitary gland, eball (including accessory glands), lung, stomach, thyroid (including parathyroid bodies), heart, bladder, and bone marrow (femur) were collected and 10% neutral phosphorus was collected. After fixation with acid-buffered formalin solution (Davidson solution for eyeball and Harder's gland), it was stored.At the end of administration, hematoxylin and eosin-stained specimens were prepared and examined microscopically in the control of anatomical animals and in the 1000 mg / kg group of male and female hearts, liver, spleen, kidney, and adrenal gland. In addition, the testis and recovery period of 1 male in the 20 mg / kg group and 2 males in the 140 mg / kg group and the testis of 1 male in the 1000 mg/ kg group at the end of the administration period, which had undergone macroscopic changes A similar study was performed on the lungs of one male in the control group and the thymus of one male in the 1000 mg / kg group at the end.
3. GROSS PATHOLOGY: Yes all rats of main and recovery groups were euthanized by over dose of carbon dioxide followed by exsanguination. The animals were examined externally in unopened condition. This was followed by opening of the carcasses and topographic examination of different organs. This included careful examination of the external surface of the body, all orifices, cranial, thoracic and visceral cavities and their contents. Simultaneously gross lesions examination was performed in accordance with the Standard Operating Procedure (SOP) of the Laboratory. Number of implatation sites in uterus and number of corpora lutea of all pregnant females were counted during necropsy examination.Similarly, necropsy of terminally sacrificed and found dead pups during study period were conducted and gross pathological observations were recorded.
HISTOPATHOLOGY: Yes Tissues were preserved in 10 % neutral buffered formalin (NBF) (except eyes, which was fixed using Modified Davidson’s fluid; testes and epididymis, which were fixed in Bouin’s fluid for approximately 24 hours and subsequently preserved in 10 % NBF) for subsequent histopathological examination. Organ examined. Adrenals, Pancreas, Aorta, Peyer's Patches, Bone (femur) with joint , Pituitary, Brain, (cerebrum,cerebellum,mid brain), Prostate and Seminal vesicle with coagulating glands as a whole, Cecum Rectum, Colon, Salivary glands Duodenum, Sciatic Nerve, Epididymide, Skeletal muscle, Eyes with optic nerve, Skin, Spinal Cord (cervical, mid-thoracic and lumbar), Heart, Spleen, Ileum, Sternum with marrow, Jejunum, Stomach, Kidneys, Testes, Liver,Thymus, Lungs, Thyroid with Parathyroids, Mammary glands, Trachea, Mesenteric and Mandibular
lymph node, Urinary Bladder, Oesophagus, Uterus, Ovaries with oviduct, Cervix with Vagina and Gross lesion (if any), were examined.
Statistics:
2. The metric data were tested for equal variances using the Bartlett method, and if the variance was uniform, a one-way analysis of variance was performed, followed by a mean comparison test using the Dunnett or Scheff method. When the variance was not uniform, the Kruskal-Wallis test was performed, and the Dunnett or Scheff type rank sum test was performed. For counting data obtained from urinalysis, Armitage's χ ^ 2 test was used. The significance level was 5% or less.
3. Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption,Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t test,Dunn’s Test, Kruskal-Wallis, ANOVA on ranks
Clinical signs:
no effects observed
Description (incidence and severity):
2. There were no deaths or abnormal findings throughout the study
3. No apparent treatment related clinical signs were observed in any of the animals throughout the treatment period. However, statistically significant increase was noted in urine pool in group G2 in male on week 2 as compared to the G1 control group. In recovery female, statistically significant decrease was noted in rearing in G4-R on pretreatment, and increase in fecal bolus on week 1 as compared to the control recovery group G1-R. Statistically significant increase in urine pool in male G2 groupon week 2 as compared to the control group G1.Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period in Main study.The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence considered as incidental and not attributed to the effect of test item administration
Mortality:
no mortality observed
Description (incidence):
2. There were no deaths or abnormal findings throughout the study
3. No mortality was observed other than two deaths due to gavage error (one male at 750 and one male and 1000 mg/kg
Body weight and weight changes:
no effects observed
Description (incidence and severity):
2. Body weight remained the same in all test substance-administered groups as well as in the control group
3. Body weight changes were restricted to a significant decrease in % body weight change in the recovery group of male rats treated at 1250 mg/kg from day 1 – 22 as compared to the control group. This effect was considered incidental and therefore not attributed to the test chemical.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
2. food consumption remained the same in all test substance-administered groups as well as in the control group
3. food intake remained unaffected by treatment.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
2. At the end of the treatment period, males in the 20 mg / kg group showed low MCHC, but not in the 140 and 1000 mg / kg groups, indicating changes not related to test substance administration. It was judged. In addition, at the end of the recovery period, a high monocyte ratio was observed in females in the 1000 mg / kg group, but not at the end of the administration period
2. Changes in haematology in the adult rats included a significant decrease in activated partial thromboplastin time in male rats treated at 1250 mg/kg compared to controls; a significant increase in red blood cell levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; a significant increase in platelet levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; and a significant increase in prothrombin time in the recovery group of male rats treated at 1250 mg/kg compared to controls. The changes in haematology were not consistent between groups and lacked histological correlates. Therefore, the changes in haematology were not considered to be of toxicological importance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
2. There were no changes in the test at the end of the treatment period. In the test at the end of the recovery period, a low level of urea nitrogen was observed in females in the 1000 mg / kg group, but this was not observed at the end of the administration period
3.Changes in clinical chemistry included decreased creatinine levels in male rats treated at ≥1000 mg/kg compared to controls; increased sodium levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased phosphorus levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased aspartate transaminase levels in the recovery groups of male and female rats treated at 1250 mg/kg compared to controls. The changes in clinical chemistry were not consistent between groups and lacked histological correlates. Therefore, the changes in clinical chemistry were not considered to be of toxicological importance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
2. In the examination of the administration period, the pH of the 1000 mg / kg group showed a change to the alkaline side. Although bilirubin was elevated in males in the 20 mg / kg group but not in the 140 and 1000 mg / kg groups, it was judged that the change was not related to test substance administration.No changes in pH were observed in the recovery period tests performed on females only. In addition, a low potassium level was observed in the 1000 mg / kg group, but it was not observed during the administration period. Therefore, the change was not related to the test substance administration
3. No effects were observed.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
3. Changes in motor activity and behaviour were restricted to a significant increase in grip strength in male rats treated at 1000 mg/kg bw/day as compared to control. This effect was considered incidental and therefore not attributed to the test chemical.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
2. At the end of the dosing period, females in the 140 mg / kg group showed a low relative weight of the adrenal gland but not in the 1000 mg / kg group, which is not related to test substance administration judged to be a change. In the test at the end of the recovery period, the relative weight of the ovary was observed in females in the 1000 mg / kg group, but this was not observed at the end of the administration period, so it was not related to test substance administration
3. There were no significant effects on either the absolute or the relative weight of the brain, adrenals,heart, liver, kidneys, spleen, thymus, thyroid with parathyroid, testes, or epididymides.
Gross pathological findings:
no effects observed
Description (incidence and severity):
2. There were no changes due to the administration of the test substance. Spontaneous changes included mild discoloration of the kidneys, kidney cysts, adrenal enlargement, testicular miniaturization, pulmonary hemorrhage, and thymic bleeding.
3. All adult animals were normal externally. Visceral findings included a case of mild splenic enlargement at 1000 mg/kg and one case of mild testicular shrinkage at 1250 mg/kg.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
2. There were no changes due to the administration of the test substance. Accidental changes in liver microgranulomas, basal changes in renal tubular epithelium, cystic expansion of renal tubules, renal mononuclear cell infiltration, hypotubule formation, lung bleeding, thymic bleeding was observed.
3. Liver: multifocal minimal to mild Neutrophil/lymphocyte infiltration (Male: G1:2/5, G4:1/5; Female:G1/5), focal to multifocal mild degeneration (Male: G1:1/5, Female: G4/5), Kidneys:focal to multifocal mild lymphocyte infiltration (Male: G1:2/5, G4:1/5; Female: G1/5, G4:1/5); Lungs: multifocal minimal to mild lymphocyte infiltration (Male: G1:1/5, G4:1/5; Female: G1/5, G4:1/5), Heart: focal to multifocal minimal lymphocyte infiltration (Male: G1:1/5; Female: G4:1/5), Spleen: multifocal mild Extramedullary haematopoiesis (Male: G1:1/5), Adrenals: multifocal mild cytoplasmic vacuolation (male: G1:1/5), accessory adrenocortical tissue (Unilateral: Male: G1:2/5, G4:2/5; Female: G1:1/5), Testes: Male: focal mild seminiferous tubule degeneration (Male: G1: 2/13, G2:2/13, G4: 2/13, G1-R: 1/5); focal minimal to mild retention of mature sperm (Male: G1: 1/13, G2:1/13, G3: 1/13, G4: 1/13); focal minimal to mild sloughing of round spermatid/pachytene spermatocyte (Male: G1: 1/13, G2:1/13).Lesions observed in liver, kidneys, lungs, heart, adrenals, spleen and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002).Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
3.Hormonal data showed no significant effects on the concentrations of T4 or TSH (male and females), testosterone (males) or oestradiol (females).
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
urinalysis
Critical effects observed:
no
Conclusions:
Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non-toxic to living organisms when dosed repeatedly via oral route. The No Observed Adverse Effect Level can be considered to be >1000 mg/kgbw/day. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.
Executive summary:

Various studies have been reviewed to determine the exact toxic dose of the test chemical when dosed repeatedly to test animals. These include in vivo experimental studies performed on rats for the test chemicals.

A 28 day repeated dose oral toxicity of the test chemical was determined in Sprague Dawley rats. The study was performed in accordance with Guidelines for 28 Day Repeat Dose Toxicity Testing for Chemicals (Japan). Male and female SD rats (Crj: CD, SPF) obtained from Charles River Japan Co., Ltd. were quarantined and acclimatized for 8 days and used for the test. Prior to the start of treatment, animals were divided into groups by weight-based stratified random sampling. The number of animals in each group was 6 males and 6 females, and for the control and high-dose groups, a recovery group of 14 males and 6 females was established. The age at the start of administration was 5 weeks for both males and females, and the body weight range was 165 to 190 g for males and 129 to 153 g for females. The dosing solution was prepared by suspending the test substance in 0.5% carboxymethylcellulose / sodium solution containing 0.1% Tween80 and refrigerated. The test substance in the administration solution was stable for at least 8 days under refrigerated storage conditions. As a result of repeated oral administration of the test substance to SD rats at doses of 500 and 1000 mg / kg for 7 days, no change in toxicity was observed in the 1000 mg / kg group. Therefore, in this study, the high dose was set at 1000 mg / kg, the upper limit of the guideline, and the medium dose was set at 140 mg / kg and the low dose was set at 20 mg / kg. The test substance was orally administered by gavage once a day for 28 days using a stomach tube. The dosing volume was 10 ml/kg and was calculated based on the body weight of the closest measurement day. The solvent was similarly administered to the control group. The treated and control animals were observed for changes in General condition observation, detailed clinical observation, body weight, food intake, urinalysis (male only), hematology examination, blood biochemistry examination, autopsy, organ examination. There were no deaths throughout the study period, there was no change in general condition, body weight, food consumption, and toxicological changes due to test substance administration were not observed in hematology, blood biochemistry, urinalysis, and pathology result. In the urinalysis, the pH of the 1000 mg / kg / day group of females showed an alkaline change during the administration period. Alkaline urine is generally found in metabolic and respiratory alkalosis, renal H + excretion due to renal failure and pyelonephritis. However, this change was within the range of the laboratory's background data, and no pathological changes were observed suggesting renal damage.Therefore, the NOAEL after repeated oral administration to rats was concluded to be at least 1000 mg / kg / day for both males and females.

This is supported by another study whose objective was to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of the test chemical. The study was performed according to OECD Guideline for the Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”; adopted: 29thJuly, 2016.A total of 124 Wistar rats (62 males and 62 females) were randomly allocated to six different dose groups for Main study. Each group consisted of 13 animals/sex for main group and 5 animals/sex for recovery group. The animals allocated to Group G2, G3, and G4/G4-R received 750, 1000 and 1250 mg/kg body weight of the test chemical respectively, whereas the animals of Group G1/G1-R received vehicle alone (Corn oil) for each day during the dosing periodby oral route. Dose formulations were prepared daily. Male rats were treated two weeks before mating and thereafter for a total of 48 dosing days. Female rats were treated two weeks before mating, during mating, during gestation and during lactation, for a total of approx. 63 dosing days. Recovery groups of male and female rats (5/sex/dose) were treated at 0 or 1250 mg/kg bw/day for 66 days in total. Animals in the recovery groups were allowed to recover for two weeks after the final dose was given. No morbidity was observed during the study period. No mortality was observed other than two deaths due to gavage error (one male at 750 and one male and 1000 mg/kg). Clinical findings were sporadic and of no biological significance. Body weight changes were restricted to a significant decrease in % body weight change in the recovery group of male rats treated at 1250 mg/kg from day 1 – 22 as compared to the control group. This effect was considered incidental and therefore not attributed to the test chemical. Food consumption was unaffected by treatment. Changes in motor activity and behaviour were restricted to a significant increase in grip strength in male rats treated at 1000 mg/kg bw/day as compared to control. This effect was considered incidental and therefore not attributed to the test chemical. Oestrous cyclicity was unaffected by treatment. All female rats showed evidence of copulation after the cohabitation/mating period. Pregnancy rates were 77, 62, 77 and 77% at 0, 750, 1000 and 1250 mg/kg, respectively. No significant effects were observed on gestation length or litter size. Likewise, no significant effects were observed on the number of live births, pup survival, pup weight or sex ratio. Four pups were cannibalized at the 750 mg/kg treatment group. All other pups at 0, 750, 1000 and 1250 mg/kg were normal externally. The internal examination of the pups revealed no abnormalities attributed to the test chemical. Microscopic examination of the pups’ thyroid and parathyroid glands in the 0 and 1250 mg/kg treatment groups revealed no abnormalities. Changes in haematology in the adult rats included a significant decrease in activated partial thromboplastin time in male rats treated at 1250 mg/kg compared to controls; a significant increase in red blood cell levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; a significant increase in platelet levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; and a significant increase in prothrombin time in the recovery group of male rats treated at 1250 mg/kg compared to controls. Changes in clinical chemistry included decreased creatinine levels in male rats treated at ≥1000 mg/kg compared to controls; increased sodium levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased phosphorus levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased aspartate transaminase levels in the recovery groups of male and female rats treated at 1250 mg/kg compared to controls. The changes in haematology and clinical chemistry were not consistent between groups and lacked histological correlates. Therefore, the changes in haematology and clinical chemistry were not considered to be of toxicological importance. Hormonal data showed no significant effects on the concentrations of T4 or TSH (male and females), testosterone (males) or oestradiol (females). There were no significant effects on either the absolute or the relative weight of the brain, adrenals, heart, liver, kidneys, spleen, thymus, thyroid with parathyroid, testes, or epididymides. All adult animals were normal externally. Visceral findings included a case of mild splenic enlargement at 1000 mg/kg and one case of mild testicular shrinkage at 1250 mg/kg. Microscopic examination revealed no treatment-related effects, that is, the incidences and types of lesions observed at 1250 mg/kg were comparable to that of the concurrent control groups. Ophthalmological examination was not performed, however, detailed clinical examinations and microscopic examination of the eyes with optic nerve (at 0 and 1250 mg/kg) did not reveal any abnormalities. NOAEL for the test chemical was established at 1250 mg/kg bw/day in male and female rats after oral gavage treatment for a period of 48 to 66 days.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non-toxic to living organisms when dosed repeatedly via oral route. The No Observed Adverse Effect Level can be considered to be >1000 mg/kgbw/day. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
Klimisch rating 1

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Endpoint conclusion
Quality of whole database:
waiver

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
exposure considerations
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated Dose Oral Toxicity:

Various studies have been reviewed to determine the exact toxic dose of the test chemical when dosed repeatedly to test animals. These include in vivo experimental studies performed on rats for the test chemicals.

A 28 day repeated dose oral toxicity of the test chemical was determined in Sprague Dawley rats. The study was performed in accordance with Guidelines for 28 Day Repeat Dose Toxicity Testing for Chemicals (Japan). Male and female SD rats (Crj: CD, SPF) obtained from Charles River Japan Co., Ltd. were quarantined and acclimatized for 8 days and used for the test. Prior to the start of treatment, animals were divided into groups by weight-based stratified random sampling. The number of animals in each group was 6 males and 6 males, and for the control and high-dose groups, a recovery group of 14 males and 6 males was established. The age at the start of administration was 5 weeks for both males and females, and the body weight range was 165 to 190 g for males and 129 to 153 g for females. The dosing solution was prepared by suspending the test substance in 0.5% carboxymethylcellulose / sodium solution containing 0.1% Tween80 and refrigerated. The test substance in the administration solution was stable for at least 8 days under refrigerated storage conditions. As a result of repeated oral administration of the test substance to SD rats at doses of 500 and 1000 mg / kg for 7 days, no change in toxicity was observed in the 1000 mg / kg group. Therefore, in this study, the high dose was set at 1000 mg / kg, the upper limit of the guideline, and the medium dose was set at 140 mg / kg and the low dose was set at 20 mg / kg. The test substance was orally administered by gavage once a day for 28 days using a stomach tube. The dosing volume was 10 ml/kg and was calculated based on the body weight of the closest measurement day. The solvent was similarly administered to the control group. The treated and control animals were observed for changes in General condition observation, detailed clinical observation, body weight, food intake, urinalysis (male only), hematology examination, blood biochemistry examination, autopsy, organ examination. There were no deaths throughout the study period, there was no change in general condition, body weight, food consumption, and toxicological changes due to test substance administration were not observed in hematology, blood biochemistry, urinalysis, and pathology result. In the urinalysis, the pH of the 1000 mg / kg / day group of females showed an alkaline change during the administration period. Alkaline urine is generally found in metabolic and respiratory alkalosis, renal H + excretion due to renal failure and pyelonephritis. However, this change was within the range of the laboratory's background data, and no pathological changes were observed suggesting renal damage.Therefore, the NOAEL after repeated oral administration to rats was concluded to be at least 1000 mg / kg / day for both males and females.

This is supported by another study whose objective was to provide evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of the test chemical. The study was performed according to OECD Guideline for the Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”; adopted: 29thJuly, 2016.A total of 124 Wistar rats (62 males and 62 females) were randomly allocated to six different dose groups for Main study. Each group consisted of 13 animals/sex for main group and 5 animals/sex for recovery group. The animals allocated to Group G2, G3, and G4/G4-R received 750, 1000 and 1250 mg/kg body weight of the test chemical respectively, whereas the animals of Group G1/G1-R received vehicle alone (Corn oil) for each day during the dosing period by oral route. Dose formulations were prepared daily. Male rats were treated two weeks before mating and thereafter for a total of 48 dosing days. Female rats were treated two weeks before mating, during mating, during gestation and during lactation, for a total of approx. 63 dosing days. Recovery groups of male and female rats (5/sex/dose) were treated at 0 or 1250 mg/kg bw/day for 66 days in total. Animals in the recovery groups were allowed to recover for two weeks after the final dose was given. No morbidity was observed during the study period. No mortality was observed other than two deaths due to gavage error (one male at 750 and one male and 1000 mg/kg). Clinical findings were sporadic and of no biological significance. Body weight changes were restricted to a significant decrease in % body weight change in the recovery group of male rats treated at 1250 mg/kg from day 1 – 22 as compared to the control group. This effect was considered incidental and therefore not attributed to the test chemical. Food consumption was unaffected by treatment. Changes in motor activity and behaviour were restricted to a significant increase in grip strength in male rats treated at 1000 mg/kg bw/day as compared to control. This effect was considered incidental and therefore not attributed to the test chemical. Oestrous cyclicity was unaffected by treatment. All female rats showed evidence of copulation after the cohabitation/mating period. Pregnancy rates were 77, 62, 77 and 77% at 0, 750, 1000 and 1250 mg/kg, respectively. No significant effects were observed on gestation length or litter size. Likewise, no significant effects were observed on the number of live births, pup survival, pup weight or sex ratio. Four pups were cannibalized at the 750 mg/kg treatment group. All other pups at 0, 750, 1000 and 1250 mg/kg were normal externally. The internal examination of the pups revealed no abnormalities attributed to the test chemical. Microscopic examination of the pups’ thyroid and parathyroid glands in the 0 and 1250 mg/kg treatment groups revealed no abnormalities. Changes in haematology in the adult rats included a significant decrease in activated partial thromboplastin time in male rats treated at 1250 mg/kg compared to controls; a significant increase in red blood cell levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; a significant increase in platelet levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; and a significant increase in prothrombin time in the recovery group of male rats treated at 1250 mg/kg compared to controls. Changes in clinical chemistry included decreased creatinine levels in male rats treated at ≥1000 mg/kg compared to controls; increased sodium levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased phosphorus levels in the recovery group of male rats treated at 1250 mg/kg compared to controls; decreased aspartate transaminase levels in the recovery groups of male and female rats treated at 1250 mg/kg compared to controls. The changes in haematology and clinical chemistry were not consistent between groups and lacked histological correlates. Therefore, the changes in haematology and clinical chemistry were not considered to be of toxicological importance. Hormonal data showed no significant effects on the concentrations of T4 or TSH (male and females), testosterone (males) or oestradiol (females). There were no significant effects on either the absolute or the relative weight of the brain, adrenals, heart, liver, kidneys, spleen, thymus, thyroid with parathyroid, testes, or epididymides. All adult animals were normal externally. Visceral findings included a case of mild splenic enlargement at 1000 mg/kg and one case of mild testicular shrinkage at 1250 mg/kg. Microscopic examination revealed no treatment-related effects, that is, the incidences and types of lesions observed at 1250 mg/kg were comparable to that of the concurrent control groups. Ophthalmological examination was not performed, however, detailed clinical examinations and microscopic examination of the eyes with optic nerve (at 0 and 1250 mg/kg) did not reveal any abnormalities. NOAEL for the test chemical was established at 1250 mg/kg bw/day in male and female rats after oral gavage treatment for a period of 48 to 66 days.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non-toxic to living organisms when dosed repeatedly via oral route. The No Observed Adverse Effect Level can be considered to be >1000 mg/kgbw/day. Comparing the above annotations with the criteria of

CLP Regulation, the test chemical can be classified under the category “Not Classified”.

Repeated Dose toxicity: Inhalation

A short-term toxicity study need not be conducted as because exposure of humans via inhalation route in production and/or use is not likely based on the provided thorough and rigorous exposure.The estimated vapour pressure of test chemical at 25 deg C was observed to be 2.08E-17 mmHg (2.78E-15 Pa). Hence, this endpoint was considered for waiver.

Repeated Dose Toxicity: Dermal

A short-term toxicity study need not be conducted as because exposure of humans via dermal route in production and/or use is not likely based on the provided thorough and rigorous exposure assessment. The acute dermal toxicity value for the test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. The substance failed to cause any dermal irritation as well sensitization. Considering this, the end point for repeated dermal toxicity is considered as waiver.

 

 

Justification for classification or non-classification

Available studies indicate that the test chemical lacks the potential to cause any toxicity when exposed repeatedly to living organisms via oral, dermal, inhalation route of exposure. Hence,comparing the above annotations with the criteria of CLP Regulations, the test chemical can be classified under the category “Not Classified”.