Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 15 January 2019 and 13 February 2019.
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Test item: PG-RAW-0004
Appearance: Liquid
Storage conditions: Refrigerated (2-7°C) protected from light
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™:WIST
Sex:
female
Details on test animals and environmental conditions:
Animal Information
Healthy nulliparous and non-pregnant female RccHan™:WIST albino rats were obtained from Envigo RMS (UK) Ltd.
The animals were allocated without conscious bias to cages within the treatment groups. They were housed in groups of one or fourrats of the same sex.
Each animal was identified uniquely within the study by tail marking. Each cage label was color-coded and was identified uniquely with the study number, dose level and animal mark.
The animals were allowed to acclimatizeto the conditions described below for at least five days before treatment. For those animals selected for this study, their body weights were in the range 154 to 169g and they were approximately eight to twelve weeks of age prior to dosing (Day 1). Thebody weight variation did not exceed ± 20% of the mean body weight of any previously treated animals.

Animal Care and Husbandry
Animals were housed inside a limited accessrodent facility (Building F21, Room 047). The facility was designed and operated to minimizethe entry of external biological and chemical agents and to minimizethe transference of such agents between rooms.
The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature and relative humidity controls were set to maintain the range of 20 to 24 °C and 40 to 70% respectively. Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study. Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours. Environmental parameters are archived with the departmental raw data.
Periodic checks were made on the number of air changes in the animal rooms. Temperature and humidity were monitored daily.
Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available to be automatically brought into operation should the public supply fail.
The cages were solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved softwood bark-free fiber bedding. Cages, food hoppers, water bottles and bedding were changed at appropriate intervals.
The animals were allowed free access to a standard rodent diet (Teklad2014C Diet), except for overnight prior to and approximately four hours after dosing. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
Each cage of animals was provided with an Aspen chew blocks or balls for environmental enrichment. Chew blocks or balls were provided throughout the study and were replaced when necessary. Each cage of animals was provided with a plastic shelter for environmental enrichment, which was replaced at the same time as the cages.
Each batch of diet was analyzed routinely by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinized and approved before any batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis were received routinely from the water supplier. Certificates of analysis were received routinely from the supplier of the chew blocks and balls. Since the results of these various analyses did not provide evidence of contamination that might have prejudiced the study, they are not presented.
No other specific contaminants that were likely to have been present in the diet or water were analyzed, as none that may have interfered with or prejudiced the outcome of the study was known.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The test item was formulated atconcentrationsof 30 and 200 mg/mL in the vehicle and administered at a volume of 10 mL/kg bodyweight.
The test item formulations were prepared on the day of dosing.
The absorption of the test item was not determined.
Determination of the homogeneity, stability and purity of the test itemor test item formulations were not undertaken as part of this study.
Detailed records of test item usage were maintained. The amount of test itemnecessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.
Doses:
300 and 2000 mg/kg
No. of animals per sex per dose:
1 female treated at 300 mg/kg. A total of 5 females treated at 2000 mg/kg.
Control animals:
no
Details on study design:
In the absence of data regarding the toxicity of the test item, 300mg/kg was chosen as the starting dose.
Two single animals were treated as follows:

Dose level (mg/kg) Concentration (mg/mL) Dose volume (mL/kg) Number of rats
Female
300 30 10 1
2000 200 10 1

In the absence of mortality or toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated as follows:

Dose level (mg/kg) Concentration (mg/mL) Dose volume (mL/kg) Number of rats
Female
2000 200 10 4
A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.

Dose Administration
The appropriate dose volume of the test item was administered to each rat by oral gavage using a plastic syringe and plastic catheter.
A record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly.
Formulations were stirred before and throughout the dosing procedure.

Serial Observations
Mortality
Cages of rats were checked at least twice daily for any mortalities.

Clinical Observations
Clinical observations will be performed on at least two occasions during the first hour following dosing (ca. thirty minutes apart) and thereafter at approximately hourly intervals for the remainder of Day 1 (for at least 4 hours). On subsequent days, animals were observed once in the morning and again at the end of the experimental day (with the exception of Day 15 - morning only). The nature and severity, where appropriate, of the clinical signs and the time were recorded at each observation.
All animals were observed for 14 days after dosing.

Body Weight
The weight of each rat was recorded on Days -1, 1 (prior to dosing), 8 and 15. Individual weekly body weight changes and group mean body weights were calculated.

Terminal Investigations
Method of Kill
Allanimals were humanely killed on Day 15 by carbon dioxide asphyxiation.

Macroscopic Pathology
All animals were subject to a macroscopic examination which consisted of opening the cranial, thoracic and abdominal cavities. The macroscopic appearance of the brain, cecum, duodenum, heart, kidneys, small and large intestine, liver, lungs and bronchi, spleen, stomach, subcutaneous tissue and urinary bladder was recorded.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
There were no deaths during the study.
Clinical signs:
Clinical signs observed in females dosed at 2000 mg/kg comprised piloerectionseen in all animals, hunched posture and/orunderactive behavior in 4/5 animals and partially closed eyelids and/orunsteady gait in 3/5 animals. These signs were first noted from approximately one or two hours after dosing. Recovery of animals, as judged by external appearance and behavior, was complete by Day 2. No clinical signs were seen in the animal dosed at 300mg/kg.
Body weight:
All animals were considered to have achieved satisfactory body weight gains throughout the study.
Gross pathology:
No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The acute oral toxicity test showed an LD50 of >2000 mg/kg bw.
Executive summary:

The acute oral toxicity of PG-RAW-0004 was assessed according to OECD guideline 420. In this study, 1 female rat was administered the substance at dose level of 300 mg/kg bw. The rats showed no mortality, clinical signs, body weight, necropsy. One female rat, followed by a group of 4 females was then treated at dose level 2000 mg/kg bw. This dose caused no mortality, clinical signs, normal increased in body weight and no abnormalities at necropsy. The acute oral LD50 for the substance in female rats was determined to be >2000 mg/kg bw.