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Description of key information

The acute oral toxicity of the test substance, FRET 08-0338, was assessed according to OECD Test Guideline 423 using an acute toxic class method. The LD50 was greater than 2000 mg/kg body weight.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 09 December 2014 and 24 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 423 using an acute toxic class method and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Animal supply, acclimatisation and allocation
Healthy nulliparous and non-pregnant female RccHan®:WIST albino rats were obtained from Harlan (UK) Ltd. The animals were allocated without conscious bias to cages within the treatment groups. They were housed in groups of up to three rats of the same sex. Each animal was identified uniquely within the study by tail marking. Each cage label was colour-coded and was identified uniquely with the study number, dose level and animal mark. The animals were allowed to acclimatise to the conditions described below for at least 5 days before treatment. For those animals selected for this study, their body weights were in the range 159 to 182 g and they were approximately eight to nine weeks of age prior to dosing (Day 1).

Animal housing, diet and water supply
Animals were housed inside a barriered rodent facility (Building F21, Room 044/045). The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the 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 19 to 23°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 fibre bedding. Cages, food hoppers, water bottles and bedding were changed at appropriate intervals.
The animals were allowed free access to a standard rodent diet (Harlan Teklad 2014C 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 block for environmental enrichment. Chew blocks 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 analysed routinely by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinised 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. 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 analysed, as none that may have interfered with or prejudiced the outcome of the study was known.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Treatment groups and doses
The dose levels for the study were chosen in compliance with the study guidelines. As no previous toxicological information was available the initial dose level was 300 mg/kg.

Formulation
The test substance was formulated at concentrations of 30 and 200 mg/mL in the vehicle and administered at a volume of 10 mL/kg body weight.The test substance formulations were prepared on the day of dosing.
The absorption of the test substance was not determined.
Determination of the homogeneity, stability and purity of the test substance or test substance formulations were not undertaken as part of this study.
Detailed records of test substance usage were maintained. The amount of test substance necessary 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.

Administration
The appropriate dose volume of the test substance 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.
Doses:
300 and 2000 mg/kg
No. of animals per sex per dose:
Six females per dose
Control animals:
no
Details on study design:
Serial observations
Mortality
Cages of rats were checked at least twice daily for any mortalities.

Clinical observations
Animals were observed soon after dosing and at frequent intervals for the remainder of Day 1. On subsequent days, surviving 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 surviving animals were observed for 14 days after dosing.

Body weight
The weight of each rat was recorded on Days 1 (prior to dosing), 8 and 15 or at death. Individual weekly body weight changes were calculated.

Necropsy and macropathology
Method of kill
All surviving animals 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, caecum, duodenum, heart, kidneys, small and large intestine, liver, lungs and bronchi, spleen, stomach, subcutaneous tissue and urinary bladder was recorded.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
Two females (An. Nos. 0047 and 0050) dosed at 2000 mg/kg were euthanized for welfare reasons on Day 1 approximately 5 or 6 hours after dosing. Clinical signs prior to death comprised unsteady gait, decreased activity, piloerection, reduced body tone, abnormally cold to touch, partially closed eyelids, irregular breathing and hunched posture seen in both decedents. Gasping was seen in one female (An. No 0050) and salivation was see in one female (An. No. 0047). These signs were seen from approximately thirty minutes after dosing. A loss in body weight was noted for one decedent. Macroscopic examination of the animals revealed congestion of the lungs and bronchi and black/brown fluid contents of the small intestine seen in both decedents. Also noted was a thick brown fluid in the stomach (An. No. 0047), clear fluid in the stomach (An. No. 0050) and yellow fluid contents of the large intestine (An. No. 0047).
Clinical signs:
Clinical signs of reaction to treatment seen in the surviving females dosed at 2000 mg/kg comprised unsteady gait and piloerection (An. Nos. 0048, 0049, 0051 and 0052). Decreased activity and hunched posture seen in three females (An. Nos. 0049, 0051 and 0052) and reduced body tone in two females (An. Nos. 0051 and 0052) and salivation (An. No. 0048). These signs were first noted approximately thirty minutes after dosing. Recovery of surviving animals, as judged by external appearance and behaviour, was complete by Day 4. No clinical signs were seen in any animal dosed at 300 mg/kg.
Body weight:
A low body weight gain was noted for one female (An. No. 0043) on Day 15. All other surviving animals were considered to have achieved satisfactory body weight gains throughout the study.
Gross pathology:
No abnormalities were revealed in any surviving animal at the macroscopic examination at study termination.

Mortality data

Dose

(mg/kg)

No of deaths

 in groups of 3

Day*

1

2 to 15

~5.5 h

~ 6 h

a

b

30

0/3

-

-

-

-

300

0/3

-

-

-

-

2000

1/3

0

1

0

0

2000

1/3

1

0

0

0

* = The day / time indicated is the time that the animal was euthanized for welfare reasons.

h = hour

a = first observation

b = second observation

- = not applicable

 

Body weight – Group mean values (g)

Group / Sex

 

Day

1

8

15

300 mg/kg F

Mean

173

199

203

SD

4.7

8.5

9.5

N

3

3

3

300 mg/kg F

Mean

180

198

206

SD

3.2

6.1

5.7

N

3

3

3

2000 mg/kg F

Mean

166

202

213

SD

7.5

7.8

5.7

N

3

3

3

2000 mg/kg F

Mean

176

204

216

SD

5.0

0.7

5.7

N

3

3

3

 

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute median lethal oral dose (LD50) to rats of FRET 08-0338 was demonstrated to be greater than 2000 mg/kg body weight.
Executive summary:

The acute oral toxicity of the test substance, FRET 08-0338, was assessed according to OECD Test Guideline 423 using an acute toxic class method. The LD50 was greater than 2000 mg/kg body weight.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of acute toxicity – oral endpoint
The study was conducted on the target substance in vivo, in an appropriate test species and according to internationally recognised guidelines

Justification for classification or non-classification

Substances can be allocated to one of four toxicity categories based on acute toxicity by the oral, dermal or inhalation route according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. Acute toxicity values are expressed as approximate LD50 or LC50 (inhalation) values. 

A test substance is classified according to one of these four toxicity categories when the acute LD50 value is ≤ 2000 mg/kg for exposure via the oral and dermal routes.

An in vivo study performed according to internationally recognised guidelines and conducted according to GLP gave an acute oral LD50 of > 2000 mg/kg and therefore the test substance is not classified for acute oral toxicity.