Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Oct - 26 Nov 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 03 Oct 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3050 Repaeted Dose 28-Day Oral Toxicity Study in Rodents
Version / remarks:
adopted Jul 2000
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
adopted 31 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Medicines and Healthcare Products Regulatory Agency, Department of Health, London, United Kingdom
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 - 25 °C in the dark
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle have been confirmed by analytical methods. Formulations were found stable for 15 days when stored refridgerated (2 to 8 °C) and for one day when stored at 15 to 25 °C.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 – 7 weeks
- Weight at study initiation: 131 – 166 g (males) and 107 – 143 g (females)
- Housing: 5 animals of the same sex per cage in polycarbonate cages with a stainless steel mesh lid, wood based bedding and environmental enrichment (aspen chew block and plastic shelters)
- Diet: Teklad Certified (2014C) Global 14% Protein Rodent Diet - Pellet, Envigo, Madison, Wisconsin, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 40 – 70
- Air supply: filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 17 Oct to 26 Nov 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared weekly and stored refrigerated at 2 to 8 °C for up to 15 days. Required amounts of the test item were weighed and approximately 50% of the final volume of vehicle was added. The formulation was magnetically stirred until the test item was uniformly mixed. Afterwards the remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. At administration, formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

VEHICLE
- Justification for use and choice of vehicle: Based on a solubility test, the test item forms uniform suspension in corn oil. Hence, corn oil was used as vehicle for dose formulation preparations.
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability of the test item in corn oil formulations was assessed at nominal concentrations of 1 mg/mL and 200 mg/mL during ambient and refrigerated storage. Samples of each formulation prepared for administration in weeks 1 and 4 of treatment were analyzed for achieved concentration of the test item. All samples were analyzed after 1, 2 and 24 hours storage at 21 °C and after 1, 8 and 15 days storage at 4°C. For each set, samples were drawn from the top, middle and bottom-layers of each preparation. The test item in the dose formulation was determined using Gas Chromatography with FID detector. The test item was used as the analytical standard. The homogeneity and stability of the test item formulation in corn oil was confirmed at nominal concentrations during distribution between bottles, during magnetic stirring for two hours, ambient temperature storage (15 to 25 °C) for 1 day and refrigerated storage (2 to 8°C) for up to 15 days. The mean concentrations of the test item in the test formulations were within +10/-15% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a foregoing dose range-finding toxicity study, in which Wistar rats were orally exposed for 14 days to 250, 500 and 1000 mg/kg bw/day Propane-1, 2, 3-Triyl 2-Ethylhexanoate. A control group was not included in this dose-range finding study. Animals receiving 500 mg/kg bw/day were younger than those reveiving 250 or 1000 mg/kg bw/day. However, when compared to the 250 mg/kg bw/day dose group, animals of the 1000 mg/kg bw/day dose group gained 13.6% less body weight after 14 days of substance administration (see Table 1 and 2 in the attached background material). Thus, it was considered appropriate to investigate a high dose level of 1000 mg/kg bw/day in this study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination and arena observations were performed on each animal during each week of treatment. Any deviation from normal was recorded with respect to nature and, where appropriate, degree of severity.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on Day 1 of treatment, weekly throughout study and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for week before treatment started and for each week throughout the study.

WATER CONSUMPTION: No
- Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food at termination
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No. 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes. Particular attention was paid to possible sings of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
- Time schedule for examinations: during Week 4 of treatment
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity (approach response, pinna reflex, auditory startle reflex, tail pinch response), grip strength and motor activity

IMMUNOLOGY: No

THYROID HORMONE ANALYSIS
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were subjected to detailed necropsy. A full macroscopic examination of the tissues listed in Table 3 was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue was recorded and tissue samples were preserved in fixative.

HISTOPATHOLOGY: Yes
For all control animals and rats treated at 1000 mg/kg bw/day, all tissue samples mentioned in Table 3 were dehydrated, embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. From animals treated at the lower dose groups 100 and 300 mg/kg bw/day only abnormalities underwent histopathological examination.

Statistics:
- Bartlett’s test for variance homogeneity
- if the Bartlett’s test was not significant at the 1% level, a F1 approximate test for parametric analysis was performed
- if the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, a William’s test for monotonic trend was applied
- if the Barlett’s test was still significant at the 1% level, a non-parametric analysis was performed following both logarithmic and square-root transformations (H1 approximate test)
- if the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley’s test for a monotonic trend was applied
- if the H1 approximate test was significant, Steel’s test was performed instead
- Fisher’s exact test for pairwise comparison of each treatment dose group against the control (grip strength, morot activity, clinical pathology)
- analysis of covariance for organ weight data using terminal body weight as covariate unless non-parametric methods were applied

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological examination did not reveal any test item-related, toxicologically significant differences from control animals. All inter-group differences from control were considered as minor as they lacked dose-response relationship. There were no changes of hematology parameters in females. Effects observed in males for different dosing groups were considered as minor as they lacked dose-response relationship and therefore were attributed to normal biological variation (non adverse).
The findings included:
- statistically significant increase in eosinophil and neutrophil counts in treated males
- statistically significant lower reticulocytes in males
- slightly high hemoglobin concentration in males treated at 300 and 1000 mg/kg bw/day
- increase of mean cell hemoglobin concentration in males at all test item concentrations
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Biochemical examination of blood plasma did not reveal any test item-related, toxicologically significant differences from control animals. Statistically significant changes were observed in males and females for different dosing groups. All inter-group differences from control were considered as minor as they lacked dose-response relationship and therefore were attributed to normal biological variation (non adverse).
The findings included:
- an increased urea concentration in females at 1000 mg/kg bw/day
- an increased triglyceride concentration in males at 100 and 1000 mg/kg bw/day
- statistically decreased potassium concentration in treated males of all dose groups compared to controls, without dose dependence; mean value of controls was noticeably higher than mean value of historical control data
- statistically significant decrease in phosphorus concentration in all treated males whithout dose dependence
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights were unaffected by treatment. Liver weights were statistically significantly increased in males at 1000 mg/kg bw/day (+9.1%). This finding was considered as minor and was attributed to normal biological variation because of its single occurence, it lacked dose-response relationship, the change was minimal, and no histopathological correlate was found.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect observed

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
It is concluded that the oral gavage administration Propane-1, 2, 3-Triyl 2-Ethylhexanoate to Han Wistar rats for 4 weeks at doses of 100, 300 or 1000 mg/kg/day was well-tolerated and did not cause any adverse change. The no-observed adverse-effect level (NOAEL) was therefore considered to be 1000 mg/kg/day of Propane-1, 2, 3-Triyl 2-Ethylhexanoate.