Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin Irritation: The substance was classified as non-irritant (OECD 439, GLP study)

Skin Corrosion: The substance was considered to be non-corrosive to the skin (OECD 431, GLP study)

Eye Irritation: The substance was considered to be non-irritating to the eye (OECD 405, GLP study)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 April 2019 - 08 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Chemical Name: 6-methyl-3,4-dihydro-2H-1,4-benzoxazine
CAS Number: 71472-57-6
Batch: 1394901004
Purity: >99% (by HPLC area % 225 nm bandwidth 50 nm)
Sponsor’s Description: Oil
Covance Description: Light yellow liquid
Expiry Date: 01 September 2019
Storage Conditions: Approximately 4°C in the dark, under nitrogen
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN™ Reconstructed Human Epidermis Model Kit
Details on animal used as source of test system:
Supplier : EpiSkin Laboratories, Lyon, France
Date received : 02 April 2019
EpiSkinTM Tissues (0.38cm2) lot number : 19-EKIN-014
Maintenance Medium lot number : 19-MAIN3-014
Assay Medium lot number : 19-ESSC-013
Justification for test system used:
Following a full validation study (ECVAM, 2009) the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
Vehicle:
unchanged (no vehicle)
Details on test system:
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells.

One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:

10 µL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.

If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.

The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and non-viable, water killed, tissues.

This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues.

Water-killed tissues were prepared prior to the study by placing untreated EPISKINTM tissues in a 12 well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5% CO2 in air for a minimum of 48 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.

In addition to the normal test procedure, the MTT reducing test item was applied to three water killed tissues. In addition, three water killed tissues remained untreated. The untreated water killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 µL of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory : Yes
Temperature Indicator Color Satisfactory : Yes
Agar Medium Color Satisfactory : Yes

2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre labeled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 C, was pipetted into the second column of 3 wells of the 12 well plate.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 Minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42 Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator determination.

2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.

For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’.
The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT 4500 microplate reader.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
83.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Direct MTT Reduction
The MTT solution containing the test item turned blue. Therefore, an assessment found the test item was able to directly reduce MTT and an additional procedure using water-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.

Assessment of Color Interference with the MTT endpoint
The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

  Quality Criteria

The relative mean tissue viability for the positive control treated tissues was4.4% relative to the negative control treated tissues and the standard deviation value of the viability was1.6%. The positive control acceptance criteria were therefore satisfied.

The mean OD570for the negative control treated tissues was0.845and the standard deviation value of the viability was3.5%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was8.3%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was classified as non-irritant. The following classification criteria apply:

EU CLP: Not classified for Irritation.
UN GHS: Not classified for Irritation (category 3 can not be determined).
Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. 

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable, water-killed, tissues were incorporated into the testing for correction purposes. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 570 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The results obtained showed that no interference due to direct reduction of MTT occurred in the main test. It was therefore considered unnecessary to use the results of the water-killed tissues, for quantitative correction of results or for reporting purposes.

The relative mean viability of the test item treated tissues was83.6% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 March 2019 - 29 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: • Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006, 18 December 2006, of the European Parliament and of the Council on REACH
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Chemical Name: 6-methyl-3,4-dihydro-2H-1,4-benzoxazine
CAS Number: 71472-57-6
Batch: 1394901004
Purity: >99% (by HPLC area % 225 nm bandwidth 50 nm)
Sponsor’s Appearance: Oil
Covance’s Appearance: Light yellow liquid
Expiry Date: 01 September 2019
Storage Conditions: Approximately 4°C in the dark
Test system:
human skin model
Source species:
other: EpiDerm™ Reconstructed Human Epidermis Model Kit
Cell type:
other: epidermal
Cell source:
other: EpiDerm™ Reconstructed Human Epidermis Model Kit
Source strain:
other: EpiDerm™ Reconstructed Human Epidermis Model Kit
Details on animal used as source of test system:
EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received : 26 March 2019
EpiDermTM Tissues (0.63cm2) lot number : 28689
Assay Medium lot number : 032119MSC
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.
Justification for test system used:
This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.
Vehicle:
unchanged (no vehicle)
Details on test system:
Application of Test Item and Rinsing
Before pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 Minute and 60 Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT-loading. Another 24 well plate was prepared for the MTT-loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT-loading plate) was dispensed into each well. The two plates were placed into the incubator until required.

After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 Minute exposure period was returned to the incubator, while the other was being dosed for the 60 Minute exposure. For the 60 Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period.

When dosing for the 60 Minute exposure period was complete, the same procedure was repeated for the 3 Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24 well plate prepared for MTT-loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated.

After the 3 Hour MTT incubation was complete, the tissue inserts were blotted and transferred to 24 well plates for formazan (reduced MTT) extraction. The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol. The plate was covered with plate sealer, to prevent isopropanol evaporation, and stood overnight at room temperature, to allow extraction to proceed.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 µL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution
Duration of treatment / exposure:
3 minutes
60 minutes
Duration of post-treatment incubation (if applicable):
Immediate observation following exposure
Number of replicates:
Two x 24-well plates
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
92.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
90
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Direct MTT Reduction
The MTT solution containing the test item turned blue. Therefore, an assessment found the test item was able to directly reduce MTT and an additional procedure using freeze-killed tissues was performed.

The results of the freeze killed tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

Assessment of Color Interference with the MTT endpoint
The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

Test Item, Positive Control Item and Negative Control Item

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

4.1

92.3

60 minute

100*

3.4

90.0

*The mean viability of the negative control tissues is set at 100%

Quality Criteria

The mean OD570for the negative control treated tissues was 1.676 for the 3‑Minute exposure period and 1.773 for the 60‑Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 3.4% relative to the negative control following the 60‑Minute exposure period. The positive control acceptance criterion was therefore satisfied.

In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. Viable cells are able toreduce MTT to formazanwhereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

4.1

92.3

60 minute

100*

3.4

90.0

*The mean viability of the negative control tissues is set at 100%

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was considered to be non-corrosive to the skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12th August 2020 - 17th August 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Prior to undertaking in-vivo irritation testing the Study Director conducted a weight-of-evidence analysis, to ensure that the in-vivo testing was sufficiently justified. This study was conducted as both in-vitro studies were inconclusive. Eye (Bovine, OECD 437): no prediction; Eye (EpiOcular Test): borderline.

For classification purposes an in vivo study was conducted.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Chemical Name: 6-methyl-3,4-dihydro-2H-1,4-benzoxazine
CAS number: 71472-57-6
Intended use: Industrial chemical
Appearance: Light yellow liquid
Storage conditions: 2 to 8°C, protected from light. Kept in storage container under protective atmosphere.
Supplier: Sponsor
Batch number: AMRI-1394906005
Expiry date: 01 September 2020
Purity: >99%
Supplier’s responsibilities: Characterisation of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Acclimatisation period: 36 to 37 weeks prior to the start of the study
- Age at start of treatment: Approx 43 to 50 weeks
- Weight at start of treatment: 2.76 to 4.60 kg
- Housing: Individually in a plastic cage with perforated floors
- Diet: 150g of a standard laboratory rabbit diet per day
- Water: Drinking water provided ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21
- Humidity (%): Relative humidity of 45-70
- Photoperiod (hrs dark/hrs light): Lighting was controlled by means of a time switch to give 12 hours of artificial light in each 24 hour period.
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.1 mL

The eyes of each animal were examined prior to installation of the test item to ensure that there was no pre-existing corneal damage, iridial inflammation or conjunctival irritation.

The dose was instilled into the right eye by pulling the lower eyelid away from the eye ball to form a cup into which the test item was dropped. The eyelids were then held together for one second before releasing. The left eye remained untreated and unexposed for control purposes.
Duration of treatment / exposure:
72 hours
Observation period (in vivo):
The animals were returned to their cages and checked at least twice during the first hour after dosing and at regular intervals throughout the day to ensure no severe injury passed unnoticed. Ocular reactions to treatment were assessed 1, 24, 48 and 72 hours after treatment.
Number of animals or in vitro replicates:
2 male rabbits
Details on study design:
SCORING SYSTEM: The classification system (Kay & Calandra, 1962) was employed on this study.

TOOL USED TO ASSESS SCORE: An ophthalmoscope or pencil beam torch was available for use to facilitate inspection of the eyes.
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 0
Reversibility:
fully reversible within:
Remarks:
24 hours
Remarks on result:
no indication of irritation
Remarks:
The highest total mean score was 10.0 occurring at the one hour observation, however this was fully reversible within 24 hours.
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Instillation of the test item gave rise to no initial pain response.

Diffuse crimson-red conjunctival appearance (Grade 2 redness), slight swelling (Grade 1 chemosis) and slight discharge (Grade 2 discharge) were apparent in both animals one hour after installation only. The treated eyes of all animals were overtly normal from 24 hours after installation.
Other effects:
There was no sign of toxicity or ill health in any rabbit during the observation period.
Interpretation of results:
GHS criteria not met
Conclusions:
The highest total mean score was 10.0 occurring at the one hour observation; accordingly under the criteria 'Kay & Calandra (1962)' the test substance was classified as 'minimally irritating' to the eye. The substance does not require labelling in accordance with European Commission regulation 1272/2008 (CLP).
Executive summary:

Summary

The purpose of this study was to assess the eye irritation potential of 6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine (an industrial chemical) to the rabbit. The method followed was that described in:

- Method B5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No. 440/2008

- OECD Test Guideline 405, 2020

Two rabbits received a single ocular installation of 0.1 mL of the test substance as supplied; the treated eyes were observed 1, 24, 48 and 72 hours after installation.

Installation of the test item gave rise to no initial pain response.

Diffuse crimson-red conjunctival appearance, slight swelling and slight discharge were apparent in the treated eyes of both animals one hour after installation only. The treated eyes of all animals were overtly normal from 24 hours after installation.

The total mean scores were assigned according to the system of 'Kay & Calandra (1962)'. The highest total mean score was ten occurring at the one hour observation; accordingly under the criteria of 'Kay & Calandra (1962)' the test substance was classified as 'minimally irritating' to the eye.

The substance does not require labelling in accordance with European Commission regulation 1272/2008 (CLP).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Skin Irritation/Corrosion:

In a GLP compliant, guideline in-vitro skin irritation test the substance was classified as non-irritant.

In a GLP compliant, guideline in-vitro corrosion irritation test the substance was classified as non-corrosive.

Eye Irritaion:

In a GLP complaint, guideline in-vivo eye irritation test the substance was classified as non-irritant.