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Administrative data

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25th April 2019 - 22nd May 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
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

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Species: Rat
Strain: Wistar Han TM
Source: Envigo RMS
Sex: Female
Other: Females were nulliparous and non-pregnant
Number of animals for the pre-test: 1 female for each selected dose level
Number of animals for the main study: 2 females at the maximum tolerated dose
Age (beginning of treatment): At least 8 weeks (beginning of treatment)
Acclimatization: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

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

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
The test item was applied evenly to an area of clipped skin equivalent to approximately 10% of the total body surface area. The site of application was covered with a gauze dressing backed with semi-occlusive surgical tape.

REMOVAL OF TEST SUBSTANCE
The exposure period was twenty-four hours, then the dressings were carefully removed. Residual test item was removed by a cotton wool tissue soaked in water to remove any residual test item.

TEST MATERIAL
Dose administration was once dermal (topical). Based on the results obtained in the concurrent oral toxicity study (Envigo Study Number 1949303, oral LD50 determined to be between 300 – 2000 mg/kg b.w.), 1000 mg/kg was chosen as the initial starting dose, since a severe toxicity which may necessitate humane euthanasia was not expected at this dose level.
Duration of exposure:
Twenty-four hours
Doses:
1000 and 2000 mg/kg
No. of animals per sex per dose:
One animal at 1000mg/kg and 3 animals at 2000mg/kg
Control animals:
no
Details on study design:
After removal of the dressings and subsequently once daily for 14 days, the test sites were examined for evidence of primary irritation and scored.

Clinical observations and inspections for morbidity / mortality were performed at least four times within the first six hours after application (i.e., 30 minutes and 1 hour, 2 hours and 4-5 hours after dosing, and in the 1st pre-experiment additionally 6 hours after dosing), thereafter at least once daily for 14 days. All animals were observed for 14 days after dosing.

Body weights were recorded on Day 0 (prior to dosing), Day 7, and 14, or (if applicable) at death (unscheduled).

At the end of the study the animals were killed by CO2 asphyxiation. A gross necropsy was performed on all animals that died or were humanely killed during the study (if applicable) and at the end of the in-life part. This consisted of an external examination and opening of the abdominal and thoracic cavities. Any macroscopic abnormalities were recorded. Routinely no organs or tissues were retained.
Statistics:
None specified

Results and discussion

Preliminary study:
No preliminary study
Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
no indication of skin irritation up to the relevant limit dose level
Mortality:
There were no deaths during the study.
Clinical signs:
In one animal, closed eyes and underactivity were observed 1 – 4 h after application. In another animal, partially closed eyes and underactivity were observed 2 h after application only. In two animals, an orange discoloration of the urine on day 1 after exposure was noted.
Body weight:
The animals showed expected gains in body weight over the observation period except for a transient minor decrease in body weight (2.2%) in animal 3 during the first week.
Gross pathology:
No abnormalities were noted at necropsy.
Other findings:
Dermal reactions included scaly skin and small scratches and small red spots. A skin erythema (score 1-2 in animal 2, score 2-4 in animal 3) was noted transiently up to day 3 after exposure.

Applicant's summary and conclusion

Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
The acute median lethal oral dose (LD50) to rats of the test substance was demonstrated to be greater than 2000 mg/kg body weight. The substance is included in Category 5/Unclassified, according to the Globally Harmonised System (GHS).
Executive summary:

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

Methods

Following a sighting test at dose levels of 1000 and 2000 mg/kg b.w. in one female rat per dose group, a further group of two females was given a single, 24 hour, semi‑occluded dermal application of the undiluted test item to intact skin at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

Results

Mortality: There were no deaths.

Clinical Observations: Closed eyes were observed in one animal treated with 2000 mg/kg b.w. from 0.5 – 4 h after application. Partially closed eyes and underactivity were noted 2 hours after application only in another animal treated with 2000 mg/kg b.w.. Additionally,a transient orange discoloration of the urine (due to the inherent colour of the test item) was observed.

Dermal Irritation: Signs of dermal reaction included scaly skin, skin erythema, small spot of eschar formation (in one animal treated with 2000 mg/kg b.w. only) and minor scratches.

Body Weight: All animals showed expected gains in body weight except for one animal treated with 2000 mg/kg b.w. which showed a transient minor decrease (-2.2%) in body weight on day 7 only.

Necropsy: No abnormalities were noted at necropsy.

Conclusion

The acute dermal median lethal dose (LD50) to rats of the test substance was found to be greater than 2000 mg/kg body weight.

The substance is included in Category 5/Unclassified according to the Globally Harmonised System (GHS).