Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2020
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Principles of method if other than guideline:
The purpose of this study was to assess the potential for systemic toxicity of pentyl propionate, when administered by oral gavage to female Sprague Dawley rats for 14 days.
Three groups, each comprising of four female Sprague Dawley rats, received pentyl propionate at doses of 500, 750 or 1000 mg/kg/day. A similarly constituted control group received the vehicle, corn oil, at the same volume dose as the treated groups.
During the study, clinical condition, body weight, food consumption, visual water consumption, organ weight and macropathology investigations were undertaken.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Test item: Pentyl propionate
Test item identity (including alternative names): Amyl propionate, 1-Pentyl propionate
CAS number: 624-54-4
Appearance: Colorless, clear liquid.
Storage conditions: At ambient temperature (15 to 25°C). Flammable liquid and vapor, therefore containers were kept tightly sealed and away from heat, sparks, open flames and hot
surfaces.
Supplier: Sponsor
Batch number D229I9D725
Recertification date: 17 April 2021
Purity: 99.96%
Specific gravity: 0.87
Appearance: Colorless, clear liquid.

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was chosen as the test species because it is accepted by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.
Sex:
female
Details on test animals and environmental conditions:
Number of animals: 20 females.
The spare animals remained in the study room throughout the treatment period. They were retained for the possibility that they might be required to form another dose group should the objective of the study not be met with the designated three dose groups.
Duration of acclimatization:
Group 1 and 2: Eight days before commencement of treatment.
Group 3 and 4: 20 days before commencement of treatment.
Age of the animals at the start of treatment:
Group 1 and 2: Approximately 73 days
Group 3 and 4: Approximately 85 days
Weight range of the animals at the start of treatment:
Group 1 and 2: 239 to 290 g
Group 3 and 4: 265 to 297 g

Animal facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Cages: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution: Females were blocked by group and the cages constituting each group were dispersed in a battery so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable.
Number of animals per cage: Two, unless reduced by mortality.
Bedding: Wood based bedding which was changed at appropriate intervals each week.

Diet: SDS VRF1, pelleted diet.
Availability: Non-restricted.

Water Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Route of Administration:
The oral gavage route of administration was chosen because it is the preferred route of exposure specified in the relevant subsequent studies test guidelines.

Rationale for Dose Level Selection:
The doses used in this study (0, 500, 750 and 1000 mg/kg/day) were selected in conjunction with the Sponsor.
The initial dose selected for dosing Group 2 animals was 500 mg/kg/day based on the results of an acute oral toxicity study in the Sprague-Dawley rat that indicate the LD50 is greater than 16 mL/kg (Meyers and Christopher, 1988), which is equivalent to greater than 13920 mg/kg based on a specific gravity of 0.87 for pentyl propionate.
After five consecutive days of dosing the Group 2 animals with pentyl propionate at 500 mg/kg/day, there were no clinical signs observed or changes in body weight, body weight gain or food consumption. Therefore, a high dose of 1000 mg/kg/day (limit dose) was selected for administration to the Group 4 animals and an intermediate dose of 750 mg/kg/day was selected for Group 3 (representing the median between the low and high dose).
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Method of preparation:
The required amount of test item was weighed out and 50% of the final volume of vehicle was added to the test item. The formulation was magnetically stirred until all the test item was uniformly mixed. Vehicle was added to achieve the final required volume and the formulation was magnetically stirred until homogenous. Magnetic stirring was continued during the transfer of the formulations, via syringe, to the final containers.

Frequency of preparation:
At least weekly. The frequency of preparation was dependent on the ongoing receipt of the stability results from Covance Study Number FC62JS. Initially the test item formulations were prepared daily but preparations in the latter part of the study covered one week of dosing.

Storage of formulation: Refrigerated temperature (2-8°C).

Vehicle: Corn oil.

DOSE ADMINISTRATION:
Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Selected doses in mg/kg/day.
Volume: dose 4 mL/kg/day.
Individual dose volume: Calculated from the most recently recorded scheduled body
weight.
Control (Group 1): Vehicle at the same volume dose as the treated groups.

Frequency: Once daily at approximately the same time each day.
Groups 2, 3 and 4 were dosed for 14 days. As a result of the staggered start of dosing, Group 1 animals were dosed for 26 days as their necropsy coincided with that of Groups 3 and 4.

Formulation: Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability: The homogeneity and stability of formulations during storage was determined as part of another study.
Formulations were shown to be stable when stored as follows:
Ambient temperature (15-25°C) for 24 hours.
Refrigerated temperature (2-8°C) for 15 days.

Achieved concentration: The dose formulation prepared for dosing to Group 2 on Days 9 to 14 of treatment was analyzed for achieved concentration. This was necessary because the dose containers for this group were stored at ambient temperature for more than 24 hours (correct storage for these dose pots
was refrigerated temperature).
The analysis was to determine if the prolonged storage at ambient temperature had any impact on the test item content in the formulation.

Analysis: Three dose containers labelled for dosing to Group 2 on 09, 10 and 11 September were sent to the Department of Dose Formulation Analysis at Covance Huntingdon and on receipt were placed in refrigerated storage (2-8°C).
The dose formulation in the dose pot labelled 11 September 2019 was analyzed. Prior to analysis the dose formulation was magnetically stirred for a minimum of 20 minutes and two 1 mL samples (accurately weighed) were taken from the middle of the formulation. Both samples were analyzed.
The analytical method involved the extraction and dilution of the test formulation with acetone followed by gas chromatographic assay using FID detection.
The residual sample and the dose containers labelled 09 and 10 September were disposed of once satisfactory results were obtained.
Duration of treatment / exposure:
14 Days
Frequency of treatment:
Once daily at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Four per dose group
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Clinical Observations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing:
Detailed observations were recorded daily at the following times in relation to dose administration:
Pre-dose.
As each animal was returned to its home cage.
One to two hours after dosing each group.
As late as possible in the working day.

Clinical Signs:
A detailed physical examination was performed on each animal three days before commencement of treatment, once during each treatment week and on Day 15 (Groups 2, 3 and 4) or Day 27 (Group 1) before dispatch to necropsy, to monitor general health.

Mortality:
A viability check was performed near the start and end of each working day.

Body Weight:
The weight of each animal was recorded three days before treatment commenced, on the day that treatment commenced (Day 1), daily throughout the study and before necropsy.

Food Consumption:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the three days before treatment started and daily throughout the study.

Water Consumption:
Fluid intake was scheduled to be assessed by daily visual observation from Day -3 until termination. For Groups 3 and 4 the observations were not undertaken in the pre-treatment phase of the study (See Section 4). No effect on water consumption was observed and consequently quantitative measurements were not performed.
Sacrifice and pathology:
Terminal Investigations:
Method of Kill:
Carbon dioxide asphyxiation with subsequent exsanguination.

Necropsy:
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.

Organ Weights:
Kidneys (weighed together) and liver were weighed for all animals killed at the scheduled intervals.

Fixation:
Tissues were preserved in 10% Neutral Buffered Formalin
Statistics:
The report contains serial observations pertaining to all days of study completed. In the case of clinical signs, body weight and food consumption data, only information from the final three days of the acclimatization period are presented.
Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data. Group mean values and standard deviations were frequently calculated using a greater number of decimal places than presented in the appendices. It is, therefore, not always possible to derive exact group values from the data presented in the appendices.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The general appearance and behaviour of all animals were unaffected by treatment. On Days 10 and 11 of treatment piloerection was seen after dosing in females receiving 1000 mg/kg/day (seen in two animals on Day 10 and four animals on Day 11).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One animal died prematurely; a female that received 500 mg/kg/day (Group 2: Animal No. 6) was found dead on Day 9 of treatment, with no prior signs recorded. The macroscopic examination revealed distension of the stomach, abnormally dark content of the duodenum, edema of the thymus gland and clear fluid in the thoracic cavity. This death was considered incidental and not related to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was an initial effect on body weight gain in females receiving 750 or 1000 mg/kg/day. In these two groups, all animals lost weight on the first day after dosing and thereafter their body weight gains were variable. This resulted in lower overall group mean weight gains in these two groups when compared with the Controls.
The body weight gains of females receiving 500 mg/kg/day were broadly similar to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption.
Food consumption in all groups was generally slightly lower during treatment compared with pre-treatment values. The difference was considered likely due to the use of corn oil as the formulation vehicle since corn oil inherently has a degree of calorific content, such that the animals require less food intake to maintain their nutritional requirements.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on kidney or liver weights at any dose level.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no adverse findings reported at the macroscopic examinations.

Effect levels

Remarks on result:
not measured/tested

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Formulation Analysis:

The dose formulation prepared for dosing to the Group 2 animals on Days 9 to 14 of treatment was stored incorrectly. Samples were subsequently taken from this formulation and analyzed for achieved concentration. The results were within acceptable limits (RME limits: maximum % 10, minimum % -15) demonstrating that the prolonged storage of the dose at ambient temperature had no impact on the integrity of the study.

Applicant's summary and conclusion

Conclusions:
Treatment of non-pregnant Sprague Dawley rats with pentyl propionate at doses up to and including 1000 mg/kg/day for 14 days was well tolerated. At dose levels of 750 and 1000 mg/kg/day slight body weight losses were seen after the first day of dosing and body weights in these groups of animals continued to fluctuate during the remainder of the treatment period, resulting in slightly lower overall body weight gains when compared to controls.
Executive summary:

The purpose of this study was to assess the potential for systemic toxicity of pentyl propionate, when administered by oral gavage to female Sprague Dawley rats for 14 days.

Three groups, each comprising of four female Sprague Dawley rats, received pentyl propionate at doses of 500, 750 or 1000 mg/kg/day. A similarly constituted control group received the vehicle, corn oil, at the same volume dose as the treated groups.

During the study, clinical condition, body weight, food consumption, visual water consumption, organ weight and macropathology investigations were undertaken.

There were no premature deaths related to treatment. One female receiving 500 mg/kg/day (Group 2: Animal No. 6) was found dead on Day 9 of treatment, with no prior signs recorded. The macroscopic examination revealed distension of the stomach, abnormally dark contents of the duodenum, edema of the thymus gland and clear fluid in the thoracic cavity; this death was considered incidental and not related to treatment.

The general appearance and behaviour of the animals were unaffected by treatment. On Days 10 and 11 of treatment piloerection was seen after dosing in females receiving 1000 mg/kg/day (seen in two animals on Day 10 and four animals on Day 11).

There was an effect on body weight gain in females receiving 750 or 1000 mg/kg/day. In these two groups all animals lost weight on the first day after dosing. Thereafter, the bodyweights of these animals fluctuated resulting in lower overall group mean weight gains

when compared to controls. The body weight gains of females receiving 500 mg/kg/day were not affected by treatment.

Food and water consumption were not affected by treatment with pentyl propionate.

There were no adverse findings at macroscopic examination and there were no kidney or liver weight effects.