Registration Dossier

Administrative data

Description of key information

Skin Irritation:

1-bromo-3-methoxybenzene (CAS No: 2398-37-0) is determined to be irritating to the skin.

Based on the observed result 1-bromo-3-methoxybenzene (CAS No: 2398-37-0) can be considered to be irritating to skin and can be classified under the category ˋ2’ as per CLP regulation.

Eye Irritation:

1-bromo-3-methoxybenzene (CAS No: 2398-37-0) was determined to be irritating to the eye.

Based on the estimated result 1-bromo-3-methoxybenzene (CAS No: 2398-37-0) can be considered to be irritating to eyes and can be classified under the category ˋ2’ as per CLP regulation.

 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 05, 2017 to July 17, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be dermal irritants. The dermal irritation potential of test article may be predicted by measurement of their cytotoxic effect, as reflected in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiDerm™ model.
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material:- Expiration date of the lot/batch:- Purity test date:RADIOLABELLING INFORMATION (Not applicable)- Radiochemical purity: N/A- Specific activity: N/A- Locations of the label: N/A- Expiration date of radiochemical substance: N/ASTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature or Fridge storage- Stability under test conditions: No data available- Solubility and stability of the test substance in the solvent/vehicle: No data available- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data availableTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: Prior to the main test, the test articles are tested for their ability to reduce/interact with MTT and their ability to stain the tissues itself. All tests are performed according to the by MatTek provided test protocol. - Preliminary purification step (if any): No data available- Final dilution of a dissolved solid, stock liquid or gel: No data available- Final preparation of a solid: No data availableFORM AS APPLIED IN THE TEST: LiquidOTHER SPECIFICS: No data available
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: as provided by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Source strain:
other: Not applicable
Details on animal used as source of test system:
- Description of the cell system used:The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native epidermis. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.Test System IdentificationAll of the EpiDerm™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues are included in this report. Tissue plates were appropriately labeled with study information.
Justification for test system used:
The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, In Vitro Life Science Laboratories, Bratislava, Slovakia) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD). Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.
Vehicle:
unchanged (no vehicle)
Details on test system:
The tissues were exposed to the test article neat (undiluted) on April 25, 2018 (Run 1 of 1). EpiDerm™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 1 hour, with 35 minutes in an approximately 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature. Following the exposure time, the tissues were rinsed and placed in fresh media for approximately 24 hours. The media was then changed again and the tissues were incubated in fresh media for another ~18 hours for a total of approximately 42 hour post-exposure recovery period. The tissue viability was then assessed by MTT assay. The tissue CoA was used instead of verification of barrier properties of the tissue.MTT and Color Pre-testsPretesting has actually been conducted for all chemicals, although the first intitial 8 test chemicals a pretesting was not conducted (for skin).MTT AssayFollowing the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 3 hours MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was approximately 3 hours with gentle shaking. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean negative control tissues.Evaluation of Test Article in the Cell Models:1. Cell system: Upon receipt, the MatTek EpiDerm™ tissue cultures were placed in 0.9 mL of fresh Maintenance medium (in a 6-well plate). The culture inserts are incubated for ~one hour. The tissues were then transferred to 6-well plates containing 0.9 mL fresh Maintenance medium and they were incubated overnight (18 ± 3 hrs) at ~37°C, 5% CO2 in a humidified incubator.2. Control and Test Article Exposures: On the day of dosing, the tissues are then removed from the incubator and the controls and the test article are applied topically to tissues by pipette(liquid) Tissues were exposed to controls and the test article for one hour, with ~35 minutes in a 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature.a) Controls30 µL of negative control DPBS and 30 μL of the positive control 5% SDS was applied topically to the tissue and gently spread by placing a nylon mesh on the apical surface of each tissue, if necessary.b) Test Articles 30 µL of the test article was applied topically to the tissue 3. Post-exposure treatmentAfter the 1 hour exposure, the tissues were rinsed 15 times with sterile DPBS. After the 15th rinse from washing bottle, each insert wasw completely submerge 3 times in 150 ml DPBS. The apical surface was gently blotted with a cotton swab. The tissues were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for 24 ± 2 hours. After this initial ~24 hour incubation, the tissues were placed in 6-well plates containing 0.9 mL fresh Maintenance medium and incubated for another 18 ± 3 hours, for a total of an approximately 42 hour post-exposure incubation.RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: The EpiDerm™ 3 dimensional human tissue model- Tissue Lot number(s): 26459- Date of initiation of testing: 6/08/2017TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°C- Temperature of post-treatment incubation (if applicable): 37°CREMOVAL OF TEST MATERIAL AND CONTROLS-Volume and number of washing steps: The test substance was rinsed from the tissues with sterile DPBS by filling and emptying the tissue insert 15 times to remove any residual test material. This was followed by completely submerge the insert 3 times in 150 ml DPBS.MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 300 µL MTT medium (1.0 mg/mL).- Incubation time: After 3 hours- Spectrophotometer: Synergy H4 spectrophotometer - Wavelength: 570 nm- Filter: No data- Filter bandwidth: No data- Linear OD range of spectrophotometer: No dataNUMBER OF REPLICATE TISSUES: 3CALCULATIONS and STATISTICAL METHODSAll data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows: MTT AssayBlanks:·        The optical density (OD) mean from all replicates for each plate (ODblank). Negative Controls (NC):·        The blank corrected value was calculated: ODNC= ODNCraw– ODblank. ·        The OD mean per NC tissue was calculated. ·        The mean OD for all tissues corresponds to 100% viability. ·        The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Positive Control (PC):·        Calculate the blank corrected value: ODPC= ODPCraw– ODblank. ·        The OD mean per PC tissue was calculated. ·        The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Tested compound :·        Calculate the blank corrected value ODTT= ODTTraw– ODblank. ·        The OD mean per tissue was calculated. ·        The viability per tissue was calculated: %TT = [ODTT/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean TT = Σ %TT / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Data Correction Procedure for MTT Interfering Compounds (if applicable)True viability = Viability of treated tissue – Interference from test article = ODtvt– ODktwhere ODkt= (mean ODtkt– mean ODukt).ODtvt= optical density of treated viable tissueODkt= optical density of killed tissuesODtkt= optical density of treated killed tissueODukt= optical density of untreated killed tissue (NC treated tissue) Data Correction Procedure for Colored Compounds (if applicable)True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt– ODvt.ODtvt= optical density of treated viable tissue incubated in MTT mediaODvt= optical density of viable tissues incubated in media alone - Evaluation of data The results of the assay was evaluated and compared to negative control. Table: Criteria for in vitro Interpretation: In VitroResults In VivoPredictionMean tissue viability ≤50% Irritant (I), R38Mean tissue viability >50% Non-irritant (NI)- Assay quality controls- Negative Controls (NC)The Dulbecco’s phosphate buffered saline (DPBS) was used as a NC. The assay passed all acceptance criteria if the ODs of the negative control exposed tissues were between ≥0.8 and ≤2.8.  - Positive Controls (PC)5% solution of sodium dodecyl sulfate was used as a PC. The assay is meeting the acceptance criteria if the viability of the PC is ≤20% of the negative control.   - Standard Deviation (SD)The standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was ≤18.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 30 µL- Concentration (if solution): neat VEHICLE (Not used)- Amount(s) applied (volume or weight with unit): none- Concentration (if solution): none- Lot/batch no. (if required): none- Purity: noneNEGATIVE CONTROL- Amount(s) applied (volume or weight): 30 µL- Concentration (if solution): neatPOSITIVE CONTROL- Amount(s) applied (volume or weight): 30 µL- Concentration (if solution): 5% solution of sodium dodecyl sulfate
Duration of treatment / exposure:
The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature.
Duration of post-treatment incubation (if applicable):
For a total of an approximately 42 hour post-exposure incubation.
Number of replicates:
3 tissues were used for test compound and control.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1
Value:
10.4
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Mean of OD:0.234;irritant
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.
Interpretation of results:
other: irritating
Conclusions:
The dermal irritation potential of test article was determined according to the OECD 439 test guideline followed for this study. The mean of OD for test chemical was determined to be 0.234.The standard deviation of viabilities for test chemical were calculated to be 0.083.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 10.4%. Thus, test chemical was considered to be irritating to the human skin.
Executive summary:

The dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met and passed the acceptance of criteria. The mean of OD  for test chemical was determined to be 0.234.The standard deviation of viabilities for test chemical were calculated to be 0.083.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 10.4%.Hence, under the current experimental test conditions it was concluded that test chemical was considered to be irritating to human skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 06, 2017 to February 16, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Principles of method if other than guideline:
The purpose of this study is to provide classification of chemicals concerning their eye irritation potential using an alternative to the Draize Rabbit Eye Test, according to the OECD Test Guideline No. 492, “Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage”. The EpiOcular™ EIT is intended to differentiate those materials that are UN GHS No Category (i.e., do not meet the requirements for UN GHS classification) from those that would require labeling as either UN GHS Category 1 or 2. This assay is not intended to differentiate between UN GHS Category 1 / Hazard
Code 318 and UN GHS Category 2 / Hazard Codes 319 and 320.
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1-bromo-3-methoxybenzene
- Molecular formula: C7H7BrO
- Molecular weight: 187.035 g/mol
- Substance type: Organic
- Physical state:liquid
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ofLOBA CHEMIE PVT. LTD.
- Lot/batch No.of test material: LB20651701
- Expiration date of the lot/batch: DEC-2D21
- Purity : 99.50%

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature / Fridge storage
- Stability under test conditions: No data available
- Solubility and stability of the test substance in the solvent/vehicle: No data available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article tested as provided neat (undiluted).
- Preliminary purification step (if any): No data available
- Final dilution of a dissolved solid, stock liquid or gel: No data available
- Final preparation of a solid: No data available

FORM AS APPLIED IN THE TEST: Solid

OTHER SPECIFICS: No data available
Species:
other: MatTek EpiOcular Tisssue Model OCL-200
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
-Test System: MatTek EpiOcular™ Tissue Model OCL-200

Storage:EpiOcular™ tissues and assay medium will be refrigerated at approximately 2-8°C upon arrival and until use.

Supplier:MatTek Corporation, Ashland, MA

- Justification of the test method and considerations regarding applicability
The EpiOcular™ Tissue Model closely parallels human ocular tissue, thus providing a useful in vitro means to assess ocular irritancy and toxicology
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl

VEHICLE (no vehicle)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none
- Lot/batch no. (if required): none
- Purity: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): neat
Duration of treatment / exposure:
Tissues will be topically exposed to the test article and control articles for 30 ± 2 minutes.
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Following the post soak,Tissues will be incubated in 1 ml fresh assay medium in a humidified 37±1°C, 5±1% CO2 incubator.




Number of animals or in vitro replicates:
2 tissues were used for test compound and control.
Details on study design:
-Plate Reader Linearity Check:
The linearity of the plate reader or spectrophotometer used for optical density (OD) determination will be verified prior to its use the same week the EIT assay is being
performed.
A dilution series of trypan blue or thiazolyl blue tetrazolium bromide (MTT) formazan will be prepared and 200 μl aliquots will be pipetted into a 96-well plate.
The optical density of the plate wells will be measured at a wavelength of 570 nm (OD570), with no reference wavelength.
A regression line and an R-squared value will be generated using Microsoft Excel®. Verification will be considered acceptable if the R-squared value is >0.999.

Assessment of Direct MTTReduction:No assessment of the direct MTT (methyl thiazole tetrazolium) reduction potential of each test article will be made.

-Assessment of Coloring or Staining Materials:
No assessment of each test article’s ability to absorb light at the wavelength (570 nm) used for MTT determination will be made.

- Pre-Incubation:
EpiOcular™ tissues will be placed in six-well plates containing warmed assay media and will be equilibrated in a humidified 37±1°C, 5 ±1% CO2 incubator for at least one hour. The media will then be changed and the tissues incubated overnight (16-24 hours).
Any tissues not being incubated the same day will be allowed to re-equilibrate at 37±1°C, 5±1% CO2 and will be stored at approximately 2-8°C..

-Pre-Treatment:After the overnight incubation, the tissues will be moistened with 20 μl of phosphatebuffered saline (PBS) and incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.

-Dosing:50 μl of a liquid test article will be applied topically to duplicate tissues and incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.
After dosing and incubation, the tissues will be thoroughly rinsed with PBS and soaked in 5 ml of room-temperature assay medium in a 12-well plate for the appropriate amount of time.
Tissues will be soaked for 12±2 minutes.

Incubation in MTT:The tissues will be incubated with 300 μl of 1 mg/ml MTT in DMEM for 3 hours ± 10 minutes at 37±1°C, 5±1% CO2

-MTT Extraction:
Following the three-hour MTT incubation period, each tissue will be removed individually and gently rinsed with PBS to remove any residual MTT solution.
The extraction plate will be covered and sealed to reduce evaporation of extractant.
For solid, colored, or staining test articles, 2.0 ml of extractant solution will be used in a six-well plate, allowing extraction to occur through the bottom of the insert.
 
Extraction Conditions:The extraction will be allowed to proceed overnight at room temperature in the dark.
Alternatively, the extraction can proceed for at least two hours, with shaking, at room temperature.

-Decant Extractant:
Tissues immersed in extractant solution in a 24-well plate: After the extraction period is complete, the liquid within each tissue insert will be decanted back into the well
from which it was taken, i.e., the solution will be mixed with the extractant in the well.
The tissue inserts will then be discarded.

-Transferring to 96-Well Plate:Two 200 μl aliquots from each well of the extraction plate(s) will be pipetted into a 96- well microtiter plate.

-Measuring Optical Density:The optical density of the extracted samples will be determined at a single wavelength of 570 nm and using eight 200 μl aliquots of the Extractant as blanks.

Calculating Percent Viability:
The percent viability of the test tissues will be determined using the following formula:
% Viability = 100 x (ODsample / ODNegative Control)

Quality Controls:
Negative Controls: The assay meets the acceptance criterion if the OD570 of the Negative Control is greater than 0.8 and less than 2.5.
Positive Controls: The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability.
Tissue Variability: The difference in viability between identically treated tissues must be less than 20%. This applies to tissues treated with the same test article as well as living and killed controls.

Ocular Irritation Potential:
An irritant is predicted if the mean relative tissue viability of two individual tissues exposed to the test substance is less than or equal to 60% of the mean viability of the
negative control-treated tissue viability.

In Vitro Result In Vivo Prediction (GHS3)
Mean tissue viability ≤ 60% Category 1 / Hazard Code 318, or
Category 2 / Hazard Codes 319 and 320
Mean tissue viability > 60% No Category (Non-Irritating)

Borderline Results:
If the test article-treated tissue viability is 60±5%, a second EIT should be performed. If the results of the second test disagree with the first, then a third test should be performed. The conclusion will be based on the agreement of two of the three tests.

Duration:The duration of the EpiOcularTM Eye Irritation Test is approximately five days.
Irritation parameter:
other: mean % tissue viability
Run / experiment:
Run 1
Value:
34.9
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
Negative Controls: The assay meets the acceptance criterion if the OD570 of the Negative Control is greater than 0.8 and less than 2.5.
Positive Controls: The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability.
Tissue Variability: The difference in viability between identically treated tissues must be less than 20%.
Interpretation of results:
other: Category 1 or 2
Conclusions:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline . The mean % tissue viability of test substance 3-bromoanisole, CAS No. 2398-37-0 was determined to be 34.9%. Thus, substance 3-bromoanisole, CAS No. 2398-37-0 was considered to be not irritating to MatTek EpiOcular Tisssue Model OCL-200
Executive summary:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to test article and controls for ~6 hours, followed by a ~25 minute post-soak and approximately 18 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay. 

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.8 to 2.5 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be below 50% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 1.99% passing the acceptance criteria.

The mean % tissue viability of test substance 3-bromoanisole, CAS No. 2398-37-0 was determined to be 34.9%.

Hence, under the experimental test conditions it was concluded that test substance 3-bromoanisole, CAS No. 2398-37-0 was considered to be irritating to the MatTek EpiOcular Tissue Model OCL-200 and being classified as “Irritating" to eyes in Category 2.”

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation:

In different studies, 1-bromo-3-methoxybenzene has been investigated for potential for dermal irritation to a greater or lesser extent. The studies are based on in vivo and in vitro experiments in rabbits along with predicted data for target chemical and its functionally similar read across substances,

The dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met and passed the acceptance of criteria. The mean of OD  for test chemical was determined to be 0.234.The standard deviation of viabilities for test chemical were calculated to be 0.083.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 10.4%.Hence, under the current experimental test conditions it was concluded that test chemical was considered to be irritating to human skin.

 

In another study, The dermal irritation potential of test article was determined according to the OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay.  The MTT data shows that the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control.The Mean % tissue viability compared to negative control (n=3) of the 3-bromoanisole, CAS No. 2398-37-0 was determined to be 29.7%. Hence, under the experimental test conditions it was concluded that test substance 3-bromoanisole, CAS No. 2398-37-0 was considered to be irritating to the human skin and being classified as or ''Irritating to skin in Category 1 or 2” as per CLP Regulation.

Administration of 4-methoxybenzyl alcohol at a dose concentration of 500mg for 24 hrs to rabbit resulted in moderate irritation which shows skin reaction as well defined erythema and slight edema.              

 

In a similar study, A skin irritation study was performed on New Zealand White rabbits according to OECD 404 Guidelines to assess the irritation potential of the test chemical. Three rabbits were exposed to 0.5 g, moistened with 60 ml Milli-U water, applied onto clipped skin for 4 hours using a semi-occlusive dressing. Observations were made 1, 24, 48 and 72 hours, and 7 and 14 days after exposure. No evidence of full thickness destruction of the skin or scar tissue was observed during the observation period, indicating that no corrosion of the skin had occurred by dermal application of the test chemical to the intact rabbit skin. Brown staining of the treated skin by the test substance was observed on all animals during the observation period, which did not hamper the scoring of the skin reaction. Four hours exposure to 0.5 g of the test chemical resulted in very slight, well defined or moderate to severe erythema and slight or moderate oedema in the treated skin-areas of the three rabbits. The skin irritation had resolved within 14 days after exposure in one animal, but remained present up to termination in the other animals. Thus from overall observations of the study it can be considered that the test chemical was caused moderate to severe irritation to rabbit skin.

Based on the available data for the target and read across substances, 3-bromoanisole can be considered to be irritating to skin.Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 2”.

 

Eye Irritation:

In different studies, 1-bromo-3-methoxybenzene has been investigated for potential for ocular irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits for target chemical and its functionally similar read across substances,

 

The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to test article and controls for ~6 hours, followed by a ~25 minute post-soak and approximately 18 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay.  The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.8 to 2.5 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be below 50% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 1.99% passing the acceptance criteria. The mean % tissue viability of test substance 3-bromoanisole, CAS No. 2398-37-0 was determined to be 34.9%. Hence, under the experimental test conditions it was concluded that test substance 3-bromoanisole, CAS No. 2398-37-0 was considered to be irritating to the MatTek EpiOcular Tissue Model OCL-200 and being classified as “Irritating" to eyes in Category 2.”

 

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met, passing the acceptance criteria. The mean % tissue viability of test substance 3-bromoanisole (CAS No.2398-37-0) was determined to be 48.5%. Hence, under the experimental test conditions it was concluded that test substance 3-bromoanisole (CAS No.2398-37-0) was considered to be irritating to the human eyes and can thus be classified as ‘’Irritating to eyes in Category 2” as per CLP Regulation

 

Acute Eye Irritation/Corrosion Study of the test chemical was performed as per OECD guideline no. 405. Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 0.1 gof test itemwas instilled in the other (treated) eye of each rabbit.The eye was observed at 1, 24, 48, 72 hour, day 7, day 14 and day 21 for all the three animals post test item instillation.Ophthalmoscope was used for scoring of eye lesions. In the initial test,100 mg of test itemwas instilled into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions; hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 100 mgof test itemwas instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. All the animals were observed till day 21 post test item instillation. Untreated eye of all the three rabbits were normal throughout the experimental period.  The following grading scores were observed in treated eye of tested rabbits.

Observation at 1 hour after instillation of test item revealed: Cornea-No ulceration or opacity was seen in all the animals; Area of Opacity-Zero inall the animals; Iris: Normal in all the animals; Conjunctivae - Some blood vessels definitely hyperaemic (injected) was seen in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in all the animals. Observation at 24 hours after instillation of test item revealed: Cornea-Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was seen in all the animals;Area of Opacity- One quarter (or less) but not zerowas seen in all the animals;Iris:Normal in all the animals;Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was seen in all the animals;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in animal no. 1 and 3 whereas obvious swelling with partial eversion of lids was seen in animal no 2. At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 40%, 50% and 40% damage in animal no. 1, 2 and 3 respectively.Observation at 48 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was seen in all the animals;Area of Opacity-One quarter (or less) but not zero was seen inall the animals;Iris:Normal in all the animals;Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible in animal no. 3;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in animal no. 1 and 2 whereas obvious swelling with partial eversion of lids in animal no. 3.Observation at 72 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was seen in all the animals;Area of Opacity-One quarter (or less) but not zero was seen inall the animals;Iris:Normal in all the animals;Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible in all the animals;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in all the animals. Observation on day 7 after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was seen in all the animals;Area of Opacity-One quarter (or less) but not zero was seen inall the animals;Iris:Normal in all the animals;Conjunctivae-Some blood vessels definitely hyperaemic (injected) was seen in animal no.1 and diffuse, crimson color; individual vessels not easily discernible was observed in animal no. 2 and 3;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in all the animals.Observation on day 14 after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible was seen in animal no. 1 and 2 whereas no ulceration or opacity was seen in animal no. 3;Area of Opacity-One quarter (or less) but not zero was seen in animals no. 1 and 2 whereas zero was seen inanimal no. 3;Iris:Normal in all the animals;Conjunctivae -Some blood vessels definitely hyperaemic (injected) was seen in all the animals;Chemosis:Some swelling above normal (includes nictitating membranes) was seen in all the animals. Observation on day 21 after instillation of test item revealed: Cornea-No ulceration or opacity in all the animals;Area of Opacity-Zero in all the animals;Iris:Normal in all the animals;Conjunctivae -Blood vessels normal in all the animals;Chemosis:No swelling (Normal) in all the animals. The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 1.00, 0.00, 2.00, 1.00; 1.00, 0.00, 2.00, 1.33, and 1.00, 0.00, 2.00, 1.33, respectively. The effects observed in all the animals were fully reversible within an observation period of 21 days.  

Hence under the experimental test conditions, the test chemical was “An Eye Irritant (Irritating to Eyes)” of New Zealand White Female rabbit eyes.

 

An eye irritation study was conducted to assess the eye irritation potential of chemical 2-Methoxybenzyl alcohol (CAS no: 612-16-8) on three rabbits The chemical was installed into the eye of each subjects at a dose of 0.1ml and later observed for ocular lesions. Each treated rabbit showed definite conjunctival irritation which persisted until day 7. Hence, the chemical 2-Methoxybenzyl alcohol (CAS no: 612-16-8) was considered to be irritating to the rabbits’ eye.

Based on the available data for the target and read across substances, 3-bromoanisole can be considered to be irritating to eyes.Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “2”.

Justification for classification or non-classification

Available studies for 3-bromoanisole suggest that it is irritating to eyes and skin. Hence, 3-bromoanisole can be classified under the category “2” as per CLP regulation