Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.08.-13.09.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
yes
Remarks:
In study plane it is stated, that the test substance will be applied in form of a suspension in water. But the test substance in water forms inhomogeneous suspension. For this reason the test substance was administered as a suspension in olive oil.
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-dimethyl-1,3-diphenylurea
EC Number:
210-283-4
EC Name:
1,3-dimethyl-1,3-diphenylurea
Cas Number:
611-92-7
Molecular formula:
C15H16N2O
IUPAC Name:
1,3-dimethyl-1,3-diphenylurea
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Species: laboratory albino rat (it is the preferred rodent species according to the guideline)
Strain: Wistar, monitored quality
Supplier: SPF breeding, VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
Sex: females
Age: 8 weeks at the time application
Acclimatisation: 12 days
Total number: 12 females
Housing: animal room with monitoring conditions – 3 animals of one sex in one plastic breeding cage
Diet: pelleted standard diet for experimental animals ad libitum
Water: drinking tap water ad libitum (quality corresponding to Regulation No. 252/2004 Czech Coll. of Law)
Microclimatic conditions:
room temperature 22 ± 3°C, permanently monitored
relative humidity 30 – 70 %, permanently monitored
light period 12-hour light/12 hour dark
Bedding: shavings of soft wood
Identification of animals: colour marks 1 - 3 on tail of animals, each cage was marked with the number of study, sex and dose of the test substance
Health condition: certificate of good health condition – from breeding farm; no signs of diseases were observed at clinical check-in, during the acclimatisation period and before the start of study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
VEHICLE
- Amount of vehicle: 1 ml/100g suspension of animal body weight
- Justification for choice of vehicle: The test substance forms in olive oil homogenous suspension.
- Lot/batch no.: 584 7204
- Purity: 100%

MAXIMUM DOSE VOLUME APPLIED: 2000 mg/kg

PREPARATION OF DOSE: Immediately before application the test substance was weighed and mixed in vehicle.

PREPARATION OF EXPERIMENTAL ANIMALS
About twenty hours before oral administration the animals were not fed, drinking water was given ad libitum. Immediately before application the animals were weighed and distributed
to groups with 3 animals.
The feed was given to animals 3 hours after application of the test substance.


CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: Test procedure with a starting dose of 50 mg/kg was selected. This level was chosen as a starting dose because there was information about toxicity of the test substance (see Confidential Test Substance Data Sheet - Reference: Journal of Pharmacology and Experimental Therapeutics. Vol. 90, Pg. 260, 1947).
Doses:
START: 50 mg/kg – 3 females (Step No.1): no deaths ► 300 mg/kg – 3 females (Step No. 2): no deaths ► 2000 mg/kg (Step No. 3) – 3 females: the death of all three females ► 300 mg/kg -3 females (Step No. 4): no deaths ► END of study
No. of animals per sex per dose:
3 animals per dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
BODY WEIGHT
before application, at th 8th day of study and at the 15th day (before euthanasia)

CLINICAL SIGHTS
After application the animals were observed individually:
-the first day: twice (30 minutes and 3 hours after application)
-the second day: twice (in the morning and in the afternoon) and daily thereafter for 14 days.

OBSERAVTION INCLUDED
changes in skin and fur, eyes, visible mucous membranes, behaviour of animals, somatomotor activity, reactions to stimuli, and presence of lacrimation, salivation and discharge from nostrils, function of respiratory, digestive and urogenital system.

MORTALITY CONTROL
- daily

PATHOLOGICAL EXAMINATION
- Gross necrospy: 15th day
- Nutritious status, body surface, body foramina, thoracic, abdominal and cranial cavity

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - < 2 000 mg/kg bw
Based on:
test mat.
Mortality:
2000 mg/kg – the death of all three females
Clinical signs:
At the dose level 2000 mg/kg the following symptoms were observed just during the first observations period (30 minutes): tachypnoea, excitation, tremor, increased reaction to external stimuli in all females. In one female at the dose level 2000 mg/kg paralysis of pelvic legs was observed.
Body weight:
No reduction of body weight was observed.
Gross pathology:
No pathologic macroscopic changes were diagnosed during pathological examination at the dose levels 50, 300 and 2000 mg/kg

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The test substance toxicity was evaluated on the basis of mortality, clinical signs of intoxication, body weight increments during the observation period and necropsy findings at the end of study.
The test substance administered at the dose of 50 and 300 mg/kg caused no death of animals. No serious clinical signs of intoxication were detected at these doses during whole study. No pathologic macroscopic changes were diagnosed during pathological examination.
The test substance administered at the dose of 2000 mg/kg caused death of all three females.
The clinical signs of intoxication such as tachypnoea, excitation, tremor and increased reaction to external stimuli were observed 30 minutes after application in all three animals. Also paralysis of pelvic legs was observed in one female 30 minutes after application. Two females were found dead after 1 hour after application and one female died 1 hour and 30 minutes after application. No pathologic macroscopic changes were diagnosed during pathological examination.

According to the study results the value of LD50 of the test substance, Methylcentralit, for female rats is in the range > 300 mg/kg to < 2000 mg/kg.
Executive summary:

The aim of the study was to investigate acute toxic effects of the test substance, Methylcentralit, after a single oral administration to Wistar Han rats.

 

The testing was performed according Method B.1 tris: Acute Oral Toxicity - Acute Toxic Class Method, Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008.

 

The test substance was administered as a suspension in olive oil, given orally via gavage to four groups of three female Wistar rats. The volume of administered solution was 1 ml/100 g body weight of animals.

 

The dosing was performed sequentially in four groups of three females: group No. 1 - first step using the starting dose of 50 mg/kgofbody weight, group No. 2 - second step using higher dose 300 mg/kg, group No. 3 – third step using the dose 2000 mg/kg and group No.4 – fourth step using lower dose 300 mg/kg.

 

The test substance administered at the dose of 50 and 300 mg/kgcaused no death of animals. No serious clinical signs of intoxication were detected during whole study. No pathologic macroscopic changes were diagnosed during pathological examination.

 

The test substance administered at the dose of 2000 mg/kg caused death of all three females.

The clinical signs of intoxication such as tachypnoea, excitation, tremor, increased reaction to external stimuli were observed immediately 30 minutes after application in all three animals. Paralysis of pelvic legs was observed 30 minutes after application only in one female. Two females were found dead 1 hour after application and one female died 1 hour and 30 minutes after application.

 

According to the study results the value of LD50of the test substance, Methylcentralit for female rats is in the range > 300 mg/kg to < 2000 mg/kg.