1,2,3-tris(2,3-epoxypropoxy)propane

Endpoint summary

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:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague-Dawley rats are widely used in toxicity studies and a large amount of historical data have been accumulated, facilitating the interpretation and evaluation of the test results.

Sex:

male/female

Details on test animals or test system and environmental conditions:

2.4.1. Animal information
Species and strain : NSam: Sprague-Dawley Rat
Microbiological grade : Specific Pathogen Free(SPF)
Breeder : Samtako Bio Korea (105, Seorang-ro, Osan-si, Gyeonggi-do, Republic of Korea)
Supplier : Young Bio Co., Ltd. (388, Dunchon-daero, Jungwon-gu, Seongnam-si, Gyeonggi-do, Republic of Korea)

Sex Male Female
Number At receipt 33 33
At first dose 30 30
Age(week) At receipt 5 5
At first dose 6 6
Body weight ranges at first dose 243.3 ~ 277.3 g 168.6 ~ 202.5 g



2.5.1. Environmental conditions
Quarantine room : Quarantine room 1-1
Test room : Animal room 14
Cage type and size : Stainless wire cage(310 W×500 L ×200 H mm)
Number of animals per cage : Less than 3 animals/cage
Temperature : Quarantine room 1-1: 21.7~22.3 ºC, Animal room 14: 21.8~22.8 ºC
Relative humidity : Quarantine room 1-1: 55.8~66.3 %, Animal room 14: 51.3~63.2 %
Air exchange : 15~18/hr
Light cycle : 12-hour light/12-hour dark (On: 8 AM, Off: 8 PM, controlled by automated timer)
Intensity of illumination : 150~300 lux
Environmental measurement : During the test period, the temperature and humidity of the animal room was recorded every 1-hour by an automatic temperature and humidity meter with a 10-minutes interval.
Environmental conditions, such as illuminance were regularly measured in accordance with “Facility Management and Operations(SOP-GER-015)” of CentralBio Co., Ltd.
Feed : Radiation-sterilized animal feed for rats, 1314 IRR[Altromin Spezialfutter GmbH & Co. KG(Im Seelenkamp 20, D-32791 Lage Postfach 11 20, D-32770 Lage_Germany)] was given adlibitum, and the contamination identification was confirmed by receiving a report from the manufacturer.
Water : Tap water was filtered and sterilized by ultraviolet sterilizer and microfiltration device and was given ad libitum in 500 mL polycarbonate water bottles. The water was periodically analyzed in accordance with “Feed and Water management(SOP-ANM-006)” of CentralBio Co., Ltd.

Route of administration:

oral: gavage

Vehicle:

DMSO

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

once a day

Dose / conc.:

88 mg/kg bw/day (nominal)

Dose / conc.:

175 mg/kg bw/day (nominal)

Dose / conc.:

350 mg/kg bw/day (nominal)

No. of animals per sex per dose:

For each dose: 10 males(5 main group, 5 recovery group), 10 females (5 main group, 5 recovery group)
(0/88/175/350 (mg/kg/day))

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

3.4. Observations
3.4.1. Clinical signs
Clinical signs were observed at least once daily, and observation of moribund or dead animals were conducted twice daily. The first day of dosing was designated as Day 1.
3.4.2. Moribund or dead animal procedure
Moribund or dead animal was not observed.
3.4.3. Detailed clinical signs
At least once prior to the first exposure, and once a week thereafter, detailed clinical observations were made in all animals. These observations were made outside the home cage, preferably in a standard arena.
Changes of skin, fur, eyes and mucous membranes
Occurrence of secretions and excretions
Autonomic activity; lacrimation, piloerection, pupil size and unusual respiratory pattern
Change in gait, posture, response to handling
Presence of clonic or tonic movements
Stereotypes; excessive grooming and repetitive circling
Bizarre behavior; self-mutilation and walking backwards
3.4.4. Sensory reactivity
All animals were examined for following items in 4th week of dosing and 2nd week of recovery.
Visual
Auditory
Touch response
Pain response
Righting reflex
3.4.5. Functional observations
Grip strength and locomotor activity were measured in 4th week of dosing(main groups) and 2nd week of recovery(recovery groups). Functional observations were omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance.
3.4.5.1. Grip strength
The front leg was measured three times using a hand-held dynamometry(BIO-GS3, Panlab, S.L.U., Spain) and averaged.
3.4.5.2. Motor activity
The distance moved was measured individually for each animal using open field test system(SuperFlex Open Field, Omnitech Electronics, Inc.). Measurements was made for five minutes and the total travel distance was used as a result.
3.4.6. Body weights
Animals were weighed on the day of animal receipt, on day of group assignment, on Day 1 (before administration), subsequently once weekly, and on the day of necropsy. After fasting overnight, body weight was measured at necropsy.
3.4.7. Food and water consumptions
Food and water consumption were measured on Day 1 and subsequently once weekly during the observation period. The remaining quantity of food and water on each following day was subtracted from the weight of food and water supplied to each cage to calculate mean daily consumption.
3.4.8. Ophthalmological examination
In 4th week of dosing(main groups) and 2nd week of recovery(recovery groups), Animals were macroscopically observed for external appearance of the eyes. Mydriatic drops(Mydriacyl, Lot. 21A25AD) were used in both eyes to facilitate mydriasis. Then anterior parts of the eye, optic media and fundus were observed with a fundus camera. No representative photographs were taken since there were no abnormal findings.


3.5. Clinical pathology
3.5.1. Urinalysis
In 4th week of dosing and 2nd week of recovery, urine was collected for 16-hours using urine plate, and chemistry examination and general examination were conduct. Urine was collected for 24-hours to obtain the total urine volume for each animal.

Chemistry examination - Multistix 10 SG (Siemens, U.S.A.) Urine analyzer (Clinitek status®, Siemens, Germany)
pH -
Specific gravity(SG) -
Protein(PRO) mg/dL
Glucose(GLU) mg/dL
Ketone body(KET) mg/dL
Bilirubin(BIL) mg/dL
Urobilinogen(URO) E.U./dL
Nitrite(NIT) -
Blood(BLO) -
Leukocyte(LEU) -

General examination
Volume mL Naked eye
Urinary sediments - Microscope
Color - Naked eye
Clarity - Naked eye

3.5.2. Blood sampling
Blood samples were collected from the posterior vena cava of all animals for scheduled necropsy under deep isoflurane anesthesia. Animals were fasted overnight (more than 16 hours) prior to sample collection.
3.5.3. Hematological test
Some blood samples were placed into CBC bottles containing EDTA as anticoagulant, and CBC(complete blood count), WBC 5 differential count and reticulocyte were analyzed using a Coulter counter(ADVIA 2120i, Siemens, Germany). The parameters are as follows:

Items Units
White blood cell count(WBC) 103 cells/μL
Differential leucocyte count
- Neutrophils(NEU)
- Lymphocytes(LYM)
- Monocytes(MONO)
- Eosinophils(EOS)
- Basophils(BASO)
103 cells/μL, %
Red blood cell count(RBC) 106 cells/μL
Hemoglobin(HGB) g/dL
Hematocrit(HCT) %
RBC indices
- Mean corpuscular volume(MCV) fL
- Mean corpuscular hemoglobin(MCH) pg
- Mean corpuscular hemoglobin concentration(MCHC) g/dL
Reticulocyte(RETI) 109 cells/L, %
Platelet(PLT) 103 cells/μL
Prothrombin time(PT) sec
Activated partial thromboplastin time(APTT) sec


3.5.4. Clinical biochemistry test
The remaining blood samples were placed into serum separating tube containing coagulation accelerator and a serum separation gel. The samples were coagulated at room temperature, and centrifuged to prepare the serum samples. The separated serum were analyzed using a biochemistry and electrolyte analyzer(Hitachi3500, HITACHI, Japan). The parameters were as follows:

Items Units
Total protein(TP) g/dL
Albumin(ALB) g/dL
Albumin/Globulin(A/G) ratio
Total bilirubin(T-BIL) mg/dL
Alkaline phosphatase(ALP) IU/L
Aspartate aminotransferase(AST) IU/L
Alanine aminotransferase(ALT) IU/L
Creatinine(CRE) mg/dL
Blood urea nitrogen(BUN) mg/dL
Total cholesterol(T-CHO) mg/dL
Triglycerides(TG) mg/dL
Glucose(GLU) mg/dL
Calcium(CA) mg/dL
Inorganic phosphorus(IP) mg/dL
Creatine phosphokinase(CK) IU/L
Sodium(Na+) mmol/L
Potassium(K+) mmol/L
Chloride(Cl-) mmol/L

3.5.5. Hormone concentration
Hormone concentration measurements were not performed because no adverse effects were observed in organ weight and histopathological examination results for the pituitary gland and thyroid gland.

3.6. Histopathology
3.6.1. Necropsy
After blood sampling, animals were sacrificed by exsanguination from the vena cava and aorta. The animals were examined carefully for external abnormalities. The abdominal, thoracic, and cranial cavities were examined for abnormalities and the organs were removed and examined.
Thyroid gland Trachea Pituitary gland
Esophagus Liver Thymus
Bone, with bone marrow+femur Adrenal gland Stomach
Kidney Lung+bronchus Pancreas
Heart+aorta Vagina Spleen
Uterus, including cervix Ovary Epididymis
Urinary bladder Testis Sciatic nerve
Skeletal muscle
Eyeball+harderian gland
Small/large intestine, including peyer’s patches
Prostate gland with coagulating gland and seminal vesicle
Brain; cerebrum, cerebellum, medulla/pons
Spinal cord, mid-thoracic
Mesenteric lymph node
Skin+mammary gland
Gross lesion
3.6.2. Organ weights measurement
The following organs were weighed and organ-to-fasted body weight ratios (relative organ weights) were calculated. The paired organs were weighed separately and then added together to calculate the total organ weight.
Thyroid gland Pituitary gland Liver
Thymus Adrenal gland Kkidney
Lung+bronchus Heart+aorta Spleen
Uterus, including cervix Ovary Epididymis
Testis
Prostate gland with coagulating gland and seminal vesicle
Brain; cerebrum, cerebellum, medulla/pons
3.6.3. Histopathological examination
3.6.3.1. Tissue fixation
The organs from all animals and organs with gross findings was preserved in 10 % neutral buffered formalin except the eyes, testis and epididymis was fixed in modified Davidson’s solution.
3.6.3.2. Slide preparation and method
Histopathological examination was included: fixed organs in all animals in vehicle control and high-dose groups, and moribund or dead animals. Tissue slides were prepared through general tissue processing and stained with Hematoxylin & Eosin (H&E). Tissues containing bones were demineralized by acid demineralization, and then slides were prepared and stained in the same way.
3.6.3.3. Histopathological examination
All prepared slides were performed to histopathological examination.

Statistics:

The data of body weight, food and water consumption, hematological test, clinical biochemistry test, and organ weights were analyzed using SPSS statistical program (IBM, Ver 25.) to compare the homogeneity of variance. The One-way ANOVA(in assumption of normality, p>0.05) was performed followed by Leven’s test for equality of variances.
In accordance with the result of Levene’s test, the Dunnett test(equal variance, p>0.05) or Dunnett T3(unequal variance, p≤0.05) was conducted as a post-hoc test to confirm the significance. In post-hoc test, p value< 0.05 is considered as statistically significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

[Main group]
Salivation was observed in all both sexes at 350 mg/kg/day on Day 26-28.
[Recovery group]
There were no test substance-related changes.

Mortality:

no mortality observed

Description (incidence):

There were no test mortalities.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

[Main group]
There were no test substance-related changes.
[Recovery group]
There were no test substance-related changes.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

[Main group]
There were no test substance-related changes.
Else, Food consumption was significantly decreased in males at 88 mg/kg/day on Day 7(P<0.05).
Food consumption was significantly decreased in females at 350 mg/kg/day on Day 1 and 14(P<0.05).
These changes were not considered to be test substance-related because there was no dose-response relationship, and/or temporary observed.
[Recovery group]
There were no test substance-related changes.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

[Main group]
There were no test substance-related changes.
[Recovery group]
There were no test substance-related changes.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

[Main group]
There were no test substance-related changes.
[Recovery group]
There were no test substance-related changes.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

[Main group]
EOS and EOS(%) in males at 350 mg/kg/day was significantly decreased(P<0.05).
EOS and EOS(%) in females at 350 mg/kg/day was decreased.
Else, PLT in male at 175 and 350 mg/kg/day were significantly increased(P<0.05). These changes were not considered to be test substance-related because there was no dose-response relationship.
[Recovery group]
EOS and EOS(%) in female at 350 mg/kg/day were significantly decreased(P<0.05).
Else, BASO(%), RBC, HGB and HCT in males at 350 mg/kg/day was decreased(P<0.05).
MCH and MCHC in females at 350 mg/kg/day were significantly increased(P<0.05).
However, there were not considered to be test substance-related because there was not relevant in the main group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

[Main group]
There were no test substance-related changes.
Else, TP in male at 300 mg/kg/day was significantly increased(P<0.05).
GLU in male at 175 and 350 mg/kg/day was significantly decreased(P<0.05).
CA in male at 175 and 350 mg/kg/day was significantly increased(P<0.05).
These changes were not considered to be test substance-related because there was not dose-response relationship, and/or not relevant changes in the other clinical chemistry items.
[Recovery group]
There were no test substance-related changes.
Else, AST, CRE and GLU in female at 350 mg/kg/day were significantly decreased(P<0.05). The changes were not considered to be test substance-related because there was not relevant change in the main groups.

Endocrine findings:

effects observed, non-treatment-related

Urinalysis findings:

no effects observed

Description (incidence and severity):

[Main group]
There were no test substance-related changes.
[Recovery group]
There were no test substance-related changes.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

[Main group]
There were no test substance-related changes.
Else, grip strength was significantly increased in male at 350 mg/kg/day(P<0.05). However, the change was not considered to be test substance-related because there was no dose-response relationship.
[Recovery group]
There were no test substance-related changes.
Else, distance moved was significantly increased in male at 350 mg/kg/day(P<0.05). However, the change was not considered to be test substance-related because distance moved was low in the vehicle control group and change was not observed in the main group.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

[Main group]
Relative weight of kidney in both sexes at 175 and 350 mg/kg/day were significantly increased(P<0.05).
Absolute weight of kidney in both sexes at 175 and 350 mg/kg/day were increased, or significantly increased(P<0.05).
Else, absolute weight of testis was significantly increased(P<0.05) and relative weight of testis was increased in male at 350 mg/kg/day.
Absolute and relative weight of lung and thyroid gland, and absolute weight of brain and uterus in females at 175 mg/kg/day were significantly increased(P<0.05).
The change was not considered to be test substance-related because there was not dose-response relationship, or not relevant changes in the histopathological examination.
[Recovery group]
Absolute weight of kidney was significantly increased(P<0.05) and relative weight of kidney was
increased in male at 350 mg/kg/day.
Absolute weight of kidney was increased and relative weight of kidney was significantly increased(P<0.05) in female at 350 mg/kg/day.
Else, absolute weight of lung in male at 350 mg/kg/day was significantly decreased(P<0.05).
Absolute and relative weight of heart in female at 350 mg/kg/day were significantly decreased(P<0.05).
These changes were not considered to be test substance-related because there was not relevant change in main group.

Gross pathological findings:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hyperplasia, limiting ridge of stomach was observed in both sexes at 350 mg/kg/day.
Else, Changes observed in the pancreas, lung, liver, thyroid gland, pituitary gland, testis and epididymis in male 0 mg/kg/day, changes observed in the kidney, lung, esophageal and thyroid gland in male 350 mg/kg/day, changes observed in the bladder, lung and thyroid gland in female 0 mg/kg/day, and changes observed in the lung, ileum and thyroid gland in female 350 mg/kg/day were not considered to be test substance-related change. Because there were background changes.
[Recovery group]
As a result of histopathological examination for the main group, no findings observed as a test substance-related change were observed, so histopathological examination was not performed on the recovery group.

Histopathological findings: neoplastic:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

not specified

Conclusions:

The purpose of this study was to confirm toxicity of KF EPIOL-PE311(GPGE) in Sprague-Dawley rats after a 28-Day repeated-dose oral administration followed by a 14-Day recovery period and determine the no-observed-adverse-effect-level (NOAEL) identify the target organ.


The test substance was administered in a 28-Day repeated-dose oral administration at a dose level of 88, 175, and 350 mg/kg/day to 5 males and 5 females per group and compared with those of the vehicle control group administered with DMSO.
Also, each 5 animals/sex were assigned to the vehicle control and high dose test-substance group in order to evaluate recovery potential during 2 weeks of recovery period.


There were no test substance-related effects in functional observations, body weights, food consumptions, water consumptions, ophthalmological examination, urinalysis and urinary sediments, clinical chemistry, and necropsy findings.


In clinical signs and detailed clinical signs, salivation observed in all both sexes at 350 mg/kg/day were considered to be test substance-related because there was observed high dose. However, the change was not considered to be toxicologically significant change because there was observed due to taste or phase of test substance.


In hematological, decreased of EOS and EOS(%) observed in both sexes at 350 mg/kg/day were
considered to be test substance-related because there was dose-response relationship or observed high dose. However, the change was not considered to be toxicologically significant change because there was recovered during the two-week recovery period or not relevant changes in the histopathological examination.


In organ weight, increased of relative and absolute of kidney observed in both sexes at 175 and 350 mg/kg/day were considered to be test substance-related because there was dose-response relationship.
However, the change was not considered to be toxicologically significant change because there were not relevant changes in the histopathological examination of kidney.


In histopathological examination, hyperplasia, limiting ridge of stomach observed in both sexes at 350 mg/kg/day were considered to be test substance-related because the change was observed due to physical simulation of test substance. However, the change was not considered to be toxicologically significant change because it was the adaptive change.


Based on the above results, KF EPIOL-PE311(GPGE) administered orally to Sprague-Dawley rats at 88, 175 and 350 mg/kg/day repeatedly for 4 weeks and a recovery period of 2 weeks, there was no toxicologically significant effect. Therefore, NOAEL of the KF EPIOL-PE311(GPGE) was determined to be 350 mg/kg/day for both males and females, and no target organ was observed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

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