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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
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

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:
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%

Test animals

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

Administration / exposure

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)).

Results and discussion

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.
Effect levels
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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).