6-methyl-3,4-dihydro-2H-1,4-benzoxazine

Endpoint summary

Description of key information

Acute Oral Toxicity (OECD 420): 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).

Acute Inhalation Toxicity (OECD 433): The LC50 (4 hour) of the test substance lies between 0.722 mg/L and 4.97 mg/L for male and female rats. The substance is classified as Category 4 according to the Globally Harmonised System (GHS; UNITED NATIONS).

Acute Dermal Toxicity (OECD 402): 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).

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:

2nd April 2019 - 24th April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Specific details on test material used for the study:

Purity: >99% (by HPLC area % 225 nm bandwidth 50 nm)

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.

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

Preliminary study:

mortality and signs of systemic toxicity at a dose level of 2000 mg/kg

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:

other: 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

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.

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).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

300 mg/kg bw

Quality of whole database:

GLP compliant guideline acute oral study with a klimisch score of 1

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August 2019 - 17 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 433 (Acute Inhalation Toxicity: Fixed Concentration Procedure)

Version / remarks:

Adopted 25 June 2018.

Deviations:

yes

Remarks:

deviations were considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Test type:

fixed concentration procedure

Limit test:

no

Specific details on test material used for the study:

Identity: 6-methyl-3,4-dihydro-2H-1,4-benzoxazine
CAS number: 71472-57-6
Intended use: Industrial chemical
Appearance: Yellow liquid
Supplier: Sponsor
Batch number: 1394901004
Expiry date: 01 January 2020
Purity: >99%

Species:

rat

Strain:

Wistar

Remarks:

RccHan®:WIST rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited (in-house)
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Sighting study: Group 1: 70 to 76 days
Main study: Group 2: 77 to 83 days
Group 3: 62 to 68 days
- Weight at study initiation:
Sighting study: Group 1: 291 g for the male;
198 g for the female
Main study: Group 2: 295 g to 316 g
Group 3: 248 g to 257 g
- Housing: one animal per cage during the sighting study and five of one sex per cage during the main study, unless this number was reduced by mortality or isolation.
- Historical data: yes
- Diet: free access to a standard rodent diet (Teklad 2014C Diet)
- Water: freely available potable water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 40 to 70%
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours continuous dark per 24 hours
- Air: filtered fresh air, which was passed to atmosphere and not re-circulated

IN-LIFE DATES:
Treatment commenced:

Sighting study Group 1 - 04 September 2019
Main study Group 2 - 18 September 2019
Group 3 - 01 October 2019


Necropsy completed:

Sighting study Group 1 - 11 September 2019
Main study Group 2 - 18 September 2019
Group 3 - 15 October 2019

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.1 - <= 2.9 µm

Geometric standard deviation (GSD):

>= 1.88 - <= 2.19

Remark on MMAD/GSD:

Group 1 - MMAD 2.1µm
GSD 1.88
Group 2 - MMAD 2.9µm
GSD 2.19
Group 3 - MMAD 2.1µm
GSD 1.92

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure system:
Flow through nose-only chamber (ADG Developments Ltd, Hitchin,Hertfordshire, England).
Aluminum alloy construction comprising a base unit, one animal exposure section with 20 exposure ports, and a top section. The dimensions of the chamber give an internal volume of approximately 30 L.
- Method of holding animals in test chamber: Plastic nose-only restraint tube.
- Source and rate of air (airflow): From in-house compressed air system – breathing quality; Generator flow: 11 L/minute.
- System of generating particulates/aerosols:
Stainless steel concentric jet atomizer (manufactured in house by Inhalation Engineering Services), designed to produce and maintain an atmosphere containing a high proportion of respirable droplets.
The test item was supplied to the atomizer, via a feed line, from a syringe (All Plastic) driven at a constant rate by a syringe pump (Harvard).
- Method of particle size determination:
Determined by cascade impaction – samples collected as follows:
Impactor type: Marple 290 Series Personal Cascade Impactor (Andersen Instruments, Smyrna, Georgia, USA)
Configuration: 298
Collection media: Stainless steel substrates and bubbler final stage
Sample flow: 2.0 L/minute
Sample volume: Measured by wet-type gas meter
Sample frequency: Two samples collected/exposure
Sample location: Animal exposure port
Sample analysis: Chemical
MMAD and GSD derived
- Treatment of exhaust air: Drawn by in-house vacuum system. Filtered locally. Flow: 12 L/minute.
- Temperature, humidity: Temperature 20.8 - 21.2°C Chamber relative humidity was not measured due to the liquid nature of the test item interfering with the test method.

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: Samples were collected using glass sintered Dreschel head and solvent trap (bubbler) containing solvent Acetonitrile, samples were injected into HPLC in duplicate
- Samples taken from breathing zone: yes. From animal exposure port

TEST ATMOSPHERE
- Particle size distribution: The mean MMAD values were within the ideal range of 1 to 4 microns, indicating the generated BN-02 aerosols were respirable to the rat.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

sighting study (group 1) - 1 mg/L
main study (group 2) - 5 mg/L
main study (group 3) - 1 mg/L

No. of animals per sex per dose:

sighting study (group 1) - 1 male; 1 female
main study (group 2) - 5 male rats
main study (group 3) - 5 male rats

Control animals:

no

Details on study design:

- Duration of observation period following administration: 7 day (sighting study) or 14 day (main study) observation period
- Frequency of observations and weighing: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants.
- Necropsy of survivors performed: yes. All main study animals were subjected to a macroscopic examination which consisted of opening the cranial, thoracic and abdominal cavities.

Detailed observations were recorded on the day of exposure, at the following times:
Pre-exposure observation
At hourly intervals thereafter, during exposure
As each animal is returned to its home cage
One and two hours after exposure
As late as possible in the working day
Observation restricted due to tube restraint.
During the observation period, surviving animals were observed once in the morning and once towards the end of the experimental day. On the day of study termination there was one observation (morning only).
Particular attention was paid to signs of evident toxicity:
Tremors, hypoactivity, irregular respiration or body weight loss (>10% compared with Day 1 prior to dosing body weight)

The weight of each sighting study animal was recorded during the week prior to exposure and on Days 1 (prior to dosing) 2, 4 and 8.
The weight of each main study animal was recorded during the week prior to exposure and on Days 1 (prior to dosing) 2, 4, 8 and at death (prior to necropsy (Day 15)).

Preliminary study:

A sighting study was conducted in one male and one female at the target concentration of 1 mg/L, which was well tolerated, and no signs of evident toxicity were seen.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

>= 0.722 - <= 4.97 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Following exposure at the mean achieved aerosol concentration of 4.97 mg/L, all five males in Group 2 (animal numbers 101-105) were killed for welfare reasonson the day of exposure (Day 1).

On return to home cage all five males were observed with a flattened posture, salivation, shallow breathing and partially closed eyes. 4/5 males were unresponsive and had a reduced body tone. One male was observed to be unsteady, had decreased activity and wet rales. One male had labored breathing; animal number 103 was also observed to be gasping and animal number 104 was cold to touch.

These signs were still present at the 1 hour after exposure check and no improvement was seen, therefore all five males were killed for welfare reasons and sent to necropsy. There were no macropathology findings observed.

Clinical signs:

irregular respiration

Body weight:

On the day following exposure for Group 1 (Day 2), body weight loss was evident in the male and body weight gain was seen in the female. Both the male and female had body weight gain at the next weighing occasion (Day 4) and at termination (Day 8).
On the day following exposure for Group 3 (Day 2) body weight loss was evident in all 5 males. The group mean body weight was comparable to Day 1 values by the next weighing occasion (Day 4) and continued to increase for the remainder of the observation period.
All changes in body weight gain were considered to be a consequence of the exposure (duration and removal of food and water) or represented normal body weight variation and therefore not test item related.

Gross pathology:

The macroscopic examination performed after a single administration followed by a 14 day observation period revealed no test-item related findings.

Other findings:

DETAILS ON CLINICAL SIGNS

There were no test item related clinical signs during the detailed weekly physical examination.
Group 1F animal number 151 exposed to the mean achieved concentration of 1.02 mg/L was found to have an unsteady gait on return to home cage, resolving by the 1 hour post exposure check, and piloerection was observed on return to home cage, resolving by the morning check on Day 2.
Group 3M animal number 106 exposed to the mean achieved concentration of 0.722 mg/L was observed to have decreased activity during the morning check on Day 2, during the afternoon check irregular breathing was also observed. Both signs had resolved by the morning check on Day 3.
No observations associated with dosing were seen in Group 1M animal number 100 exposed to the mean concentration of 1.02 mg/L or the Group 3M animal numbers 107-110 exposed to the mean achieved concentration of 0.722 mg/L.
Clinical signs associated with the dosing procedure included wet fur and staining of the nose for some animals, and this was considered to be a consequence of tube restraint during exposure.

Chamber Atmosphere Conditions

Summary data are presented below:

Group	Target aerosol level (mg/L)	Mean achieved aerosol concentration (mg/L)	Particle size
			MMAD (mm)	sg
1	1	1.02	2.1	1.88
2	5	4.97	2.9	2.19
3	1	0.722	2.1	1.92
MMAD Mass median aerodynamic diameter sg          Geometric standard deviation

The mean achieved aerosol concentration values were 102 and 99% for Groups 1 and 2, respectively. The mean achieved aerosol concentration value was 72% for Group 3, the exposure settings used for Groups 1 and 3 were the same, reasons for the lower achieved aerosol concentrations are unknown.

The mean MMAD values were within the ideal range of 1 to 4 microns, indicating the generated BN-02 aerosols were respirable to the rat.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

6-methyl-3,4-dihydro-2H-1,4-benzoxazine was administered to RccHan®:WIST rats by a nose-only, single exposure for 4 hours, followed by either a 7 day (sighting study) or 14 day (main study) observation period. Mortality was induced by humane sacrifice in all 5 males exposed to the achieved concentration of 4.97 mg/L, therefore based on this and the 2 exposures in which the animals survived; 1 male and 1 female exposed to the achieved concentration of 1.02 mg/L and 5 males exposed to the achieved concentration of 0.722 mg/L, the LC50 (4 hour) of the test substance lies between 0.722 mg/L and 4.97 mg/L for male and female rats. The substance is classified as Category 4 according to the Globally Harmonised System (GHS; UNITED NATIONS).

Executive summary:

The study was designed to investigate the acute inhalation toxicity of the test item and, if appropriate, allow the use of serial steps of Fixed Concentration Procedure (FCP) to provide a ranking of test item toxicity. Data from this study was primarily used to classify and label the test item in accordance with the United Nations (UN) Globally Harmonized

System of Classification and Labelling of Chemicals (GHS).

Test item	6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine
Test animals	RccHan^®:WIST rats
Study structure	1 sighting study, consisting of 1 male and 1 female 2 main study groups, each consisting of 5 males
Route of administration	Inhalation
Duration of exposure	Single 4-hour nose only exposure
Observation period	Sighting study group, seven days post exposure Main study groups, fourteen days post exposure

Results

A sighting study was conducted in one male and one female at the target concentration of
1 mg/L, which was well tolerated, and no signs of evident toxicity were seen. The initial group of main study animals were exposed to a target concentration of 5 mg/L, during which all five males were killed for welfare reasons. The second group of main study animals were exposed to a target concentration of 1 mg/L, all five males survived, and evident toxicity was seen during the observation period.

Exposure Levels

The mean average concentration data are summarised as follows:

Group	Target aerosol level (mg/L)	Mean achieved aerosol concentration (mg/L)	Particle size
			MMAD (mm)	sg
1	1	1.02	2.1	1.88
2	5	4.97	2.9	2.19
3	1	0.722	2.1	1.92
MMAD Mass median aerodynamic diameter sg          Geometric standard deviation

The mean achieved aerosol concentration values were 102, 99 and 72% for Groups 1, 2 and 3, respectively. The mean MMAD values were within the ideal range of 1 to 4 microns, indicating the generated substance aerosols were respirable to the rat.

Mortality

Following exposure at the mean achieved aerosol concentration of 4.97 mg/L, all five males in Group 2 (animal numbers 101-105) were killed for welfare reasons on the day of
exposure (Day 1).

 

On return to home cage all five males were observed with a flattened posture, salivation, shallow breathing and closed eyes. 4/5 males were unresponsive and had a reduced body tone. One male was observed to be unsteady, had decreased activity and wet rales. 2/5 males had labored breathing; animal number 103 was also observed to be gasping and animal number 104 was cold to touch.

 

These signs were still present at the 1 hour after exposure check and no improvement was seen, therefore all five males were killed for welfare reasons and sent to necropsy. There were no macropathology findings observed.

Clinical Signs

There were no test item related clinical signs during the detailed weekly physical examination.

Group 1F animal number 151 exposed to the mean achieved concentration of 1.02 mg/L was found to have an unsteady gait on return to home cage, resolving by the 1 hour post exposure check, and piloerection was observed on return to home cage, resolving by the AM check on Day 2.

Group 3M animal number 106 exposed to the mean achieved concentration of 0.722 mg/L was observed to have decreased activity during the AM check on Day 2, during the PM check irregular breathing was also observed. Both signs had resolved by the AM check on Day 3.

Clinical signs associated with the dosing procedure included wet fur and staining of the nose for some animals, and this was considered to be a consequence of tube restraint during exposure.

Body Weight

On the day following exposure for Group 1 (Day 2), body weight loss was evident in the male and body weight gain was seen in the female. Both the male and female had body weight gain at the next weighing occasion (Day 4) and at termination (Day 8). On the day following exposure for Group 3 (Day 2), body weight loss was evident in all 5 males. The group mean body weight was comparable to Day 1 values by the next weighing occasion (Day 4) and continued to increase for the remainder of the observation period.

Macropathology

The macroscopic examination performed after a single administration followed by a 14 day observation period revelated no test-item related findings.

Conclusion

The test substance was administered to RccHan®:WIST rats by a nose-only, single exposure for 4 hours, followed by either a 7 day (sighting study) or 14 day (main study) observation period. Mortality was induced by humane sacrifice in all 5 males exposed to the achieved concentration of 4.97 mg/L, therefore based on this and the 2 exposures in which the animals survived; 1 male and 1 female exposed to the achieved concentration of 1.02 mg/L and 5 males exposed to the achieved concentration of 0.722 mg/L, the LC₅₀(4 hour) of BN-02 lies between 0.722 mg/L and 4.97 mg/L for male and female rats. The substance is classified as Category 4 according to the Globally Harmonised System (GHS; UNITED NATIONS).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

0.722 mg/m³ air

Quality of whole database:

GLP compliant guideline acute oral study with a klimisch score of 1

Acute toxicity: via dermal route

Link to relevant study records

Reference

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

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

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.

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

Preliminary study:

No preliminary study

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:

other: 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

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.

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

GLP compliant guideline acute oral study with a klimisch score of 1

Additional information

Acute toxicity studies to OECD protocols and to acceptable quality standards are available for the oral, inhalation and dermal exposure routes. 

Justification for selection of acute toxicity – oral endpoint
Only one study available

Justification for selection of acute toxicity – inhalation endpoint
Only one study available

Justification for selection of acute toxicity – dermal endpoint

Only one study available

Justification for classification or non-classification

Acute Oral Toxicity:

In a GLP compliant, guideline acute oral toxicity test 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).

Acute Inhalation Toxicity

In a GLP compliant, guideline acute inhalation toxicity test the test substance was administered to RccHan®:WIST rats by a nose-only, single exposure for 4 hours, followed by either a 7 day (sighting study) or 14 day (main study) observation period.  Mortality was induced by humane sacrifice in all 5 males exposed to the achieved concentration of 4.97 mg/L, therefore based on this and the 2 exposures in which the animals survived; 1 male and 1 female exposed to the achieved concentration of 1.02 mg/L and 5 males exposed to the achieved concentration of 0.722 mg/L, the LC50 (4 hour) of the substance lies between 0.722 mg/L and 4.97 mg/L for male and female rats. The substance is classified as Category 4 according to the Globally Harmonised System (GHS; UNITED NATIONS).

Acute Dermal Toxicity

In a GLP compliant, guideline acute dermal toxicity test 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).