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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2nd April 2019 - 24th April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
GLP compliance:
yes (incl. QA statement)
Test type:
fixed dose procedure
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
6-methyl-3,4-dihydro-2H-1,4-benzoxazine
EC Number:
837-106-9
Cas Number:
71472-57-6
Molecular formula:
C9H11NO
IUPAC Name:
6-methyl-3,4-dihydro-2H-1,4-benzoxazine
Test material form:
liquid
Specific details on test material used for the study:
Purity: >99% (by HPLC area % 225 nm bandwidth 50 nm)

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst / The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 12 weeks
- Weight at study initiation: 166.9 - 187.1g
- Fasting period before study: The animals were fasted over night before treatment, with constant ad libitum access to drinking water.
- Housing: groups of one to five rats (of the same sex and dose group)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days prior to the start of dosing under test conditions after health examination

ENVIRONMENTAL CONDITIONS
temperature 22 + 2°C
relative humidity approx. 45-65
(with the aim of 50 – 60%)
artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 30mg/mL (300 mg/kg)
- Amount of vehicle (if gavage): The volume administered did not exceed 10 mL/kg b.w
Doses:
300 and 2000 mg/kg
No. of animals per sex per dose:
1 at each selected dose level in prelim study (300 and 2000 mg/kg)
4 at the maximum tolerated dose (300 mg/kg)
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: at least three times within the first six hours after application (i.e., 30 minutes and 1 hour, 2 hours and 4 hours after dosing), thereafter at least once daily for 14 days.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight
Statistics:
Not specified

Results and discussion

Preliminary study:
mortality and signs of systemic toxicity at a dose level of 2000 mg/kg
Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
>= 300 - <= 2 000 mg/kg bw
Based on:
test mat.
Mortality:
There were no deaths during the study at 300mg/kg.
The female (2000m/kg) used in the sighting test had to be humanely sacrificed an hour after treatment.
Clinical signs:
300 mg/kg b.w:
Closed eyes to partly closed eyes, hunched posture, lachrymation, piloerection, decreased activity, and staining of the urine (orange) were noted within 6 hours of dosing in the animals treated at a dose level of 300 mg/kg.These signs were first noted 30 minutes after dosing. Recovery of surviving animals, as judged by external appearance and behavior, was complete by Day 1.

2000 mg/kg b.w:
Clinical signs prior to sacrifice comprised closed eyes to partially closed eyes, lachrymation, lethargy, apathy, decreased respiration rate, and prostration. Additionally, the animal was moribund and unconscious.
Body weight:
The animals showed expected gains in body weight over the observation period.
Gross pathology:
Macroscopic examination at study termination on Day 14 revealed the spleen of animal 4 was swollen. The stomach of animal 1 and 4 smelled sourly. The thymus of animal 3 was speckled red. No abnormalities were noted in animals 2 and 5 at the macroscopic examination.

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The acute median lethal oral dose (LD50) to rats of the test substance was demonstrated to be between 300 and 2000 mg/kg body weight.
The substance is included in Category 4, according to the Globally Harmonised System (GHS).
Executive summary:

The study was performed to assess the acute oral toxicity of 6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine to the rat.

Methods

Following two sighting tests at dose levels 300 mg/kg b.w. and 2000 mg/kg b.w. in one female rat per dose group, a further group of fourfasted females was given a single oral dose of the test substance as a solution in corn oil at a dose level of 300 mg/kg body weight. Clinical signs and body weight development were monitored in all animals during the study. All animals were subjected to gross necropsy.

Results

Mortality: There were no deaths at dose level 300 mg/kg b.w.. The animal of the sighting test at 2000 mg/kg b.w., was humanely sacrificed 1h after the application.

Clinical Observations: Closed eyes or partially closed eyes, hunched posture, lachrymation, piloerection, decreased activity, hypothermia, and orange staining of the urine were noted within 6 hours of dosing in the animals treated at a dose level of 300 mg/kg. 
In the one animal treated at 2000 mg/kg b.w. closed eyes to partially closed eyes, lachrymation, apathy, decreased respiration rate, and prostration were observed within the first hour after dosing. Additionally, the animal was moribund and unconscious. Therefore, the animal was humanely sacrificed.

Body Weight: All animals showed expected gains in body weight.

Necropsy: The spleen of animal 4 was swollen. The stomach of animal 1 and 4 smelled sourly. The thymus of animal 3 was speckled red.

Conclusion

The acute median lethal oral dose (LD50) to rats of the test substance was demonstrated to be between 300 and 2000 mg/kg body weight.

The substance is included in Category 4 according to the Globally Harmonised System (GHS).