Registration Dossier

Administrative data

Description of key information

Two studies were performed to predict the skin irritation and corrosivity potential of the test material Vetimoss (2-tert-butyl-1,4-dimethoxybenzene, EC 244 -216 -5, CAS 21112-37-8). The in vitro skin corrosivity and irritation test EPISKIN TM (SM) was used. This system is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The testing followed OECD TGs 431 and 439 and GLP conditions.

 

Two studies were conducted to assess and evaluate the eye irritation potential of the test material. The isolated chicken’s eyes in vitro eye irritation test was used. The study was conducted according to the OECD 438 Guideline and under GLP conditions. Additionally, an in vitro eye irritation test using Reconstructed Human Cornea-like Epithelium (RhCE) was conducted. The study was conducted under GLP and in accordance with OECD Guideline 492 recommendations.

 

SKIN IRRITATION

The study was conducted according the OECD Guideline 431 and performed under GLP conditions. The test systems EPISKIN TM (SM) were treated with the test item and incubated for 15 minutes at room temperature. At the end of the exposure time, the disks were rinsed with Phosphate Buffered Saline (PBS) and incubated at 37°C for 42 hours in an incubator with 5% CO2, in a > 95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere. PBS was used as negative control and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution was used as positive control. Two additional disks were used to provide in each case an estimate of colour contribution from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. For irritation, if the mean relative viability after 15 minutes of exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

The results of the irritation test showed that the mean cell viability was 77.8% compared to the negative control. The validity criteria were acceptable. In conclusion, the test material Vetimoss (2-tert-butyl-1,4-dimethoxybenzene) was considered to be non- irritant, according to the result of this study.

 

SKIN CORROSION

The study was conducted according the OECD Guideline 439 and performed under GLP conditions. The test systems EPISKIN TM (SM) were treated with the test item and incubated for for 4 hours at room temperature. At the end of the exposure time, the disks were rinsed with Phosphate Buffered Saline (PBS) and incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere. Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid were used as negative and positive control, respectively.

Two additional disks were used to provide in each case an estimate of colour contribution from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity test, if the mean relative viability after 4 hours of exposure is <35% the test item is considered to be corrosive to skin.

The results of the corrosivity test indicated that the mean cell viability was 112.2% compared to the negative control. In conclusion, the test material Vetimoss (2-tert-butyl-1,4-dimethoxybenzene) was considered non-corrosive, according to the result of this study.

 

EYE IRRITATION

The test was performed using isolated chicken’s eyes in vitro eye irritation test. The study was conducted according to the OECD 438 Guideline and under GLP conditions. The selected eyes were held in a horizontal position and the test item was applied onto the centre of the cornea in order to cover the surface of the cornea. The cornea was exposed to the test item for 10 seconds. Then the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 µL of the undiluted test item and three positive control eyes were treated in a similar way with 30 µL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). To evaluate the irritation potential, the corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects were observed and recorded. The negative control and positive control results were within the historical control data range in the experiment. The experiment was considered to be valid. 

The result of the study showed no significant corneal swelling during the four-hour observation period post exposure. Slight cornea opacity change (severity 0.5 on one eye and severity 1.0 on two eyes) was observed in three eyes. Slight fluorescein retention change (severity 0.5 on two eyes and 1.0 on one eye) was recorded in all three eyes. However, no other corneal effects were observed. Based on the result of this study, the further testing is needed for the classification of the test item Vetimoss (2-tert-butyl-1,4-dimethoxybenzene).

A Key in vitro study (Citoxlab, 2019, K1), was conducted to predict the acute eye irritation potential of the test material (2-tert-butyl-1,4 -dimethoxybenzene) by measurement of its cytotoxic effect on the EpiOcularTM cornea epithelial model, in accordance with OECD Test Guideline No. 492 (25 June 2018). Preliminary tests were performed to detect the ability of the test material to directly reduce MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) as well as its colouring potential. Following the preliminary tests, the eye irritation potential of the test material was assessed in the main test. In the preliminary tests, the test material was neither found to have direct MTT reducing properties, nor colouring potential. In the main test, all acceptance criteria were fulfilled and the study was therefore considered to be valid. The relative mean viability of the tissues treated with the test item was 98% with a difference of 8% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.

Under the experimental conditions of this study, the test material (2-tert-butyl-1,4-dimethoxybenzene), was considered to be non-irritant to Reconstructed human Cornea-like Epithelium. Based on the results of this study, the test material should not be classified for ocular irritation under GHS (2017) and EU Regulation (EC) No. 1272/2008.

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:
From 06 June 2018 to 08 June 2018
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:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiSkin(TM) SOP, INVITTOX Protocol No 18
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Episkin(TM) SOP, Version 1.8
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test Material: Vetimoss, (2-tert-butyl-1,4-dimethoxybenzene)
- CAS Number: 21112-37-8
- EC Number: 244-216-5
- Source and lot/batch No.of test material: A180118C
- Expiration date of the lot/batch: 06 June 2019
- Purity test date: 99.78 %
- Appearance: Clear, almost colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions: Room temperature (15-25 °C, ≤ 70% relative humidity (RH)),
under inert gas, protected from humidity (tightly closed container)
- Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat)
for unknown materials were applied to assure personnel health and safety.

PREPARATION OF THE TEST ITEM
- The test item was applied as supplied, no formulation was required.


Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: NA
Source strain:
other: NA
Details on animal used as source of test system:
TEST SYSTEM: Human Skin
EPISKIN(TM) (SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-023, Expiry Date: 11 June 2018) is a three-dimensional human epidermis model. Adult humanderived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Justification for test system used:
The EPISKIN(TM) (SM) model has been validated for corrosivity and irritation testing in an international validation studies and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM (SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-023, Expiry Date: 11 June 2018)
- Units: EPISKIN TM (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm*2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
- Plate: 12-well assay plates
- Punch: EPISKIN TM (SM) biopsy punch for easy sampling of epidermis
- A flask of sterile “Maintenance Medium” (Batch No.: 18 MAIN3 028; Exp. Date: 13 June 2018); a flask of sterile “Assay Medium” (Batch No.: 18 ESSC 024; Exp. Date: 13 June 2018)
- Storage: The kits were kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.

MTT SOLUTION
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solutions were stored in refrigerator (2-8 °C) protected from light. These were diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

ACIDIFIED ISOPROPANOL
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

CHEMICALS USED IN THE EXPERIMENT:
- MTT
- Isopropanol
- Hydrochloric acid
- Phosphate buffered saline (PBS)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
General validity criteria
- The mean OD value of the two or three negative control tissues should be ≥ 0.6 and ≤ 1.5.
- The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.
Specific criteria for corrosivity
- The difference of viability between the two tissue replicates should not exceed 30%.
- The acceptable mean percentage viability range for positive controls is ≤ 20%.


Control samples:
other: Negative controls - Corrosion test: Physiological saline (0.9% (w/v) NaCl solution) - Irritation test: Phosphate Buffered Saline Positive controls - Corrosion test: Glacial acetic acid - Irritation test: 5% (w/v) Sodium Dodecyl Sulphate solution
Amount/concentration applied:
TEST MATERIAL
The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath.
50 µL of test item was applied evenly to each of two test units and each additional control skin units.

NEGATIVE AND POSITIVE CONTROL S
50 µL of negative control (Physiological saline) or positive control (glacial acetic acid) were added to each skin unit by using a suitable pipette.

Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 4 hours at room temperature (21.9-23.4°C).
Number of replicates:
TEST ITEM TEST AND CONTROLS:
- Two replicates per time point were used for test item
- Two negative controls and two positive controls
- Two additional test item-treated living tissues were used for the non-specific OD evaluation

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
97.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No indication of skin corrosion
Remarks:
Mean experiments 1,2: 112.2%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
126.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No indication of skin corrosion
Remarks:
Mean experiments 1,2: 112.2%
Other effects / acceptance of results:
General validity criteria:
- After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions.
- The mean OD value of the two negative control tissues in the corrosivity test was in the recommended range (0.804).
- The mean OD value of the blank samples (acidified isopropanol) in the corrosivity test was 0.048.

Specific criteria for corrosivity testing:
- The two positive control treated tissues showed 0.3% viability demonstrating the proper performance of the assay.
- The difference of viability between the two test item-treated tissue samples in the MTT assay was 25.4%.
- The difference of viability between the two negative control tissue samples in the MTT assay was 0.5%.

Indicator for potential false viability
- Chemical action by the test material on MTT may mimic that of cellular metabolism leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. MTT reduction and interferences were checked and corrected.




INTERPRETATION OF THE TEST RESULTS

Corrosivity test:

The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).

For 2 disks:

- Mean viability of ≥ 35% = Non Corrosive

- Mean viability of < 35% = Corrosive

Interpretation of results:
GHS criteria not met
Conclusions:
The results of the corrosivity test indicated that the mean cell viability was 112.2% compared to the negative control. The validity criteria were acceptable.
In conclusion, the test material Vetimoss, (2-tert-butyl-1,4-dimethoxybenzene) was considered non-corrosive, according to the result of this study.
Executive summary:

The aim of the study was to predict the corrosivity potential of the test material Vetimoss, (2-tert-butyl-1,4-dimethoxybenzene) using the in vitro skin irritation corrosivity test EPISKIN TM (SM), which is designed to predict and classify the corrosivity and irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

The study was conducted according the OECD Guideline 439 and performed under GLP conditions.

The test systems EPISKIN TM (SM) were treated with the test item and incubated for for 4 hours at room temperature. At the end of the exposure time, the disks were rinsed with Phosphate Buffered Saline (PBS) and incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere.

 

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid were used as negative and positive control, respectively.

Two additional disks were used to provide in each case an estimate of colour contribution from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity test, if the mean relative viability after 4 hours of exposure is <35% the test item is considered to be corrosive to skin.

The results of the corrosivity test indicated that the mean cell viability was 112.2% compared to the negative control.

In conclusion, the test material Vetimoss, (2-tert-butyl-1,4-dimethoxybenzene) was considered non-corrosive, according to the result of this study.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06 June 2018 to 08 June 2018
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)
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Episkin(TM) (SM) SOP, Version 1.0
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Episkin(TM) (SM) SOP, INVITTOX Protocol No 118
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test Material: Vetimoss, (2-tert-butyl-1,4-dimethoxybenzene)
- CAS Number: 21112-37-8
- EC Number: 244-216-5
- Source and lot/batch No.of test material: A180118C
- Expiration date of the lot/batch: 06 June 2019
- Purity test date: 99.78 %
- Appearance: Clear, almost colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions: Room temperature (15-25 °C, ≤ 70% relative humidity (RH)), under inert gas, protected from humidity (tightly closed container)
- Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.

PREPARATION OF THE TEST ITEM
- The test item was applied as supplied, no formulation was required.


Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: NA
Source strain:
other: NA
Details on animal used as source of test system:
TEST SYSTEM: Human Skin
EPISKIN(TM) (SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-023, Expiry Date: 11 June 2018) is a three-dimensional human epidermis model. Adult humanderived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Justification for test system used:
The EPISKIN(TM) (SM) model has been validated for corrosivity and irritation testing in an international validation studies and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM (SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-023, Expiry Date: 11 June 2018)
- Units: EPISKIN TM (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm*2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
- Plate: 12-well assay plates
- Punch: EPISKIN TM (SM) biopsy punch for easy sampling of epidermis
- A flask of sterile “Maintenance Medium” (Batch No.: 18 MAIN3 028; Exp. Date: 13 June 2018); a flask of sterile “Assay Medium” (Batch No.: 18 ESSC 024; Exp. Date: 13 June 2018)
- Storage: The kits were kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.

MTT SOLUTION
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solutions were stored in refrigerator (2-8 °C) protected from light. These were diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

ACIDIFIED ISOPROPANOL
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

CHEMICALS USED IN THE EXPERIMENT:
- MTT
- Isopropanol
- Hydrochloric acid
- Phosphate buffered saline (PBS)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
General validity criteria
- The mean OD value of the two or three negative control tissues should be ≥ 0.6 and ≤ 1.5.
- The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.
Specific criteria for irritation test
- Standard deviation value (SD) of the negative control tissues and the identically treated replicates % viability should be ≤ 18.
- The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be ≤ 18.

Control samples:
other: NEGATIVE CONTROLS: Phosphate Buffered Saline; POSITIVE CONTROL: 5% (w/v) Sodium Dodecyl Sulphate solution
Amount/concentration applied:
TEST MATERIAL
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath.

10 µL of test item were applied evenly to each of three test units and each additional control skin units. The amount was sufficient to cover the epidermal surface.

NEGATIVE AND POSITIVE CONTROL S
50 µL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette.

Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature (23.1-23.6°C).
Duration of post-treatment incubation (if applicable):
After rinsing, the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (+18 minutes) at 37°C in an incubator with 5% CO2, in a > 95% humidified atmosphere.
Number of replicates:
TEST ITEM TEST AND CONTROLS:
- Three replicates per time point
- Three replicates per time point were used for test item
- Three negative controls and three positive controls
- Two additional test item-treated living tissues were used for the non-specific OD evaluation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
78.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Mean experiments 1,2,3: 77.8%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
78.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Mean experiments 1,2,3: 77.8%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
75.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Mean experiments 1,2,3: 77.8%
Other effects / acceptance of results:
General validity criteria:
- After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions.
- The mean OD value of the two negative control tissues was in the recommended range (0.676).
- The mean OD value of the blank samples (acidified isopropanol) was 0.047.

Specific criteria for irritation testing:
- Standard deviation of the viability results for negative control samples was 0.2%.
- Positive control treated tissues showed 8.9% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 2.7%.
- The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 1.6%.
- All these parameters were within acceptable limits and therefore the study was considered to be valid.

Indicator for potential false viability
- Chemical action by the test material on MTT may mimic that of cellular metabolism leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. MTT reduction and interferences were checked and corrected.




INTERPRETATION OF THE TEST RESULTS

Irritation test:

- Mean tissue viability % is ≤ 50 % = Category 2 or Category 1

- Mean tissue viability % is > 50 % = Non- Irritant

Interpretation of results:
GHS criteria not met
Conclusions:
The results of the irritation test showed that the mean cell viability was 77.8% compared to the negative control. The validity criteria were acceptable.
In conclusion, the test material Vetimoss, (2-tert-butyl-1,4-dimethoxybenzene) was considered non- irritant, according to the result of this study.
Executive summary:

The aim of the study was to predict the irritation potential of the test material Vetimoss, (2-tert-butyl-1,4-dimethoxybenzene) using the in vitro skin corrosivity and irritation test EPISKIN TM (SM), which is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

The study was conducted according the OECD Guideline 431 and performed under GLP conditions.

The test systems EPISKIN TM (SM) were treated with the test item and incubated for 15 minutes at room temperature. At the end of the exposure time, the disks were rinsed with Phosphate Buffered Saline (PBS) and incubated at 37°C for 42 hours in an incubator with 5% CO2, in a > 95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere. PBS was used as negative control and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution was used as positive control.

Two additional disks were used to provide in each case an estimate of colour contribution from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. For irritation, if the mean relative viability after 15 minutes of exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

The results of the irritation test showed that the mean cell viability was 77.8% compared to the negative control. The validity criteria were acceptable.

In conclusion, the test material Vetimoss, (2-tert-butyl-1,4-dimethoxybenzene) was considered to be non- irritant, according to the result of this study.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-10-18 to 2018-10-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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)
Deviations:
yes
Remarks:
Deviation were considered not to have any impact on the validity or integrity of the study
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Kalama Chemical Ltd (United Kingdom); Batch no. A180118C
- Expiration date of the lot/batch: 2019-06-06
- Purity test date: 2018-02-07

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from humidity under nitrogen atmosphere
- Stability under test conditions: Not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material) : The test item was used in its original form and was noted as a colourless liquid.
Species:
human
Strain:
other: Reconstructed human Cornea-like Epithelium (tissues)
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
Species: Reconstructed human Cornea-like Epithelium (tissues).

Supplier: MatTek, Bratislava, Slovak Republic.

Batch No.: 27076.

Selection: at receipt, the tissues were inspected for obvious defects prior to use.

Storage conditions: As the tissues were shipped the day prior the treatment, tissues were stored between +2°C and +8°C, prior to their pre-incubation. Then tissues were equilibrated (in the 24-well shipping container) to room temperature for at least 15 minutes. Each tissue was inspected as specified in internal procedure, removed from the 24‐well shipping containers and placed in a 6-well plate containing 1 mL of pre-warmed assay medium. Tissues were then incubated for 1 hour at +37°C, 5% CO2 in a humidified incubator. After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24h) at +37°C, 5% CO2 in a humidified incubator. Each 6-well plate was labeled with the test item or control codes.

Description: the EpiOcularTM model consists of an airlifted, living, multi-layered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinized epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The 3D tissue consists of highly organized cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.

Expiry date: the EpiOcular tissues were used within 72 hours of their production.
Vehicle:
unchanged (no vehicle)
Remarks:
The test item was used in its original form
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Since the test item and both negative and positive controls could be sampled using a pipette, a volume of 50 µL of each item was evenly applied to the surface of each dedicated tissue, using a positive displacement pipette and taking care to spread the test item over the whole tissue surface area without damaging the tissue sample.
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature to remove any remaining test item from the tissue, blotted on absorbent material, and then incubated for another 2 hours at 37°C, 5% CO2 in a humidified incubator.
Number of animals or in vitro replicates:
Test item, negative and positive controls were applied on duplicate tissues.
Details on study design:
- Details of the test procedure used

- RhCE tissue construct used, including batch number : Reconstructed human Cornea-like Epithelium (MatTek, Bratislava, Slovak Republic Batch # 27076)

- Doses of test chemical and control substances used: 50 µL

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : Treatment at +37°C for 30 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature to remove any remaining test item from the tissue, blotted on
absorbent material, and then incubated for another 2 hours at 37°C, 5% CO2 in a humidified incubator.

- Description of any modifications to the test procedure : Description of test procedure/s is provided in 'Any other information on materials and methods incl. tables'.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : To identify any test substance interference with the MTT endpoint, a preliminary test was performed. If the MTT solution colour turns blue/purple when compared to the negative control, the test item was presumed to reduce MTT and additional controls were performed on freeze-dead tissues in parallel to the main test, to evaluate the part of OD due to the non-specific reduction of the MTT.
Otherwise, no additional tissue controls were used.

As a test item may be coloured or become coloured in contact with water and/or isopropanol, it is necessary to test its potential interference with the MTT determination in these two conditions. The ability of the test item to absorb significantly light at the wavelength used for MTT determination was tested. If, after subtraction of the blank OD, the OD of the test item solution was > 0.08 (approximately 5% of the mean viability of the negative control) the test item was considered as possibly interacting with the MTT measurement and additional controls were performed on viable tissues in parallel to the main test. Otherwise, no additional tissue controls were used.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : Test item, negative and positive controls were applied on duplicate tissues.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : The OD was measured at 570 nm using Multiskan GO plate reader(Brand: Thermo Scientific, Type: 1510, Serial No.: 1510.0761C.

- Description of the method used to quantify MTT formazan : provided in 'Any other information on materials and methods incl. tables'

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : The results of the study were considered acceptable if the following criteria are fully met:
• the mean OD blank of each plate (i.e. extraction solvent) is < 0.1,
• the mean cOD of the negative controls is between 0.8 and 2.5,
• relative mean viability of the positive control is < 50% of the relative mean viability of the negative control,
• the difference of viability between the two tissue replicates is < 20%.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : Not specified

- Complete supporting information for the specific RhCE tissue construct used: Yes, Certificate of Analysis of the EpiOcularTM tissues appended to the study report.

- Reference to historical data of the RhCE tissue construct : not specified

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : The Eye Irritation Test (EIT) protocol has been validated to differentiate irritant from non-irritant chemicals using the relative viability percentage. The design of this study was based on theOECD Test Guideline No. 492 (25 June 2018)and according to the protocol published in 2015 by MatTek Corporation: "EpiOcularTM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals".

- Positive and negative control means and acceptance ranges based on historical data : Not specified

- Acceptable variability between tissue replicates for positive and negative controls: The results of the study were considered acceptable if the mean cOD of the negative controls is between 0.8 and 2.5 and relative mean viability of the positive control is < 50% of the relative mean viability of the negative control.

- Acceptable variability between tissue replicates for the test chemical: The results of the study were considered acceptable if the difference of viability between the two tissue replicates is < 20%.
Irritation parameter:
other: Mean tissue viability
Run / experiment:
MTT Test
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Mean tissue viability for the negative control was 100%
Positive controls validity:
valid
Remarks:
Mean tissue viability for the positive control was 35%
Remarks on result:
no indication of irritation
Remarks:
Mean tissue viability for the test material was 98%
Other effects / acceptance of results:
Preliminary Tests
Test for direct MTT reduction potential of the test item
The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on freeze-dead tissues in parallel to the main test.

Test for colouring potential of the test item
During this test, and after subtraction of the blank OD, the OD of each water and isopropanol solutions containing the test item was ≤ 0.08 (i.e.-0.0005, 0.0005, 0.0455 and 0.0465, respectively). The test item was assessed as having no colouring potential, and therefore no interaction with the MTT measurement was considered. As a result, no additional controls were performed on viable tissues in parallel to the main test for the evaluation of the non-specific OD.

Main Test
Evaluation of the colouration of tissues at the end of the MTT incubation period: All test item-treated tissues appeared blue which was considered indicative for viable tissues.

The relative mean viability of the tissues treated with the test item was 98% with a difference of 8% between duplicate tissues.

All of the acceptance criteria were fulfilled, the study was therefore considered to be valid. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response. Moreover, a single experiment was sufficient for the test item since the resulting classification is unequivocal.

Main Test

Evaluation of the colouration of tissues at the end of the MTT incubation period:

The qualitative evaluation of the MTT staining was performed with the naked eye and results are presented in Table 1.

 

Table 1: Qualitative assessment of tissue viability

Treatment

Tissue 1

Tissue 2

Negative control

B

B

Positive control

B/W b

B/W b

Test Material

B

B

 

B: blue discolouration of the tissue

B/W: blue/white discolouration of the tissue

b: blue colouration at the periphery of tissues and white discolouration at the center of tissues

Events on tissues:

Tissue observation was performed at the rinsing and biopsy steps, and any events that occurred were reported in Table 2.

 

Table 2: Events reported on tissues

Treatment

No. Tissues

Phase

Event

All Tissues

-

-

-

-: no abnormality noted

 

No events were reported at any phase of the study.

Evaluation of the MTT results:

The individual and mean OD values, standard deviations and viabilities for the test item, negative control and positive control tissues are presented in Tables 3 and 4.

 

Table 3: Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

Group

Exposure

Duration

Tissue No.

OD570 nm measurements

Mean Blank

cOD570 nm measurements

Mean cOD570 nm

Viability (%)

1st

2nd

1st

2nd

Negative control

30 min

1

2.065

2.056

0.039

2.026

2.017

2.022

101

2

2.027

2.005

1.988

1.966

1.977

99

Positive control

30 min

1

0.758

0.756

0.039

0.719

0.717

0.718

36

2

0.739

0.738

0.700

0.699

0.700

35

Test

Material

30 min

1

2.101

2.077

0.040

2.061

2.037

2.049

102

2

1.926

1.920

1.886

1.880

1.993

94

OD = optical density

cOD = blank corrected optical density

 

Table 4: Mean tissue viability and standard deviations for the test item, the negative and positive controls

Group

Exposure

Duration

cOD570 nm

Viability (%)

Mean

SD

Mean

SD

Difference (%)

Negative control

30 min

2.000

0.031

100

2

2

Positive control

30 min

0.709

0.013

35

1

1

Test

Material

30 min

1.966

0.117

98

6

8

cOD = blank corrected optical density

SD = standard deviation

 

The relative mean viability of the tissues treated with the test item was 98% with a difference of 8% between duplicate tissues.

Interpretation of results:
other: Not irritating
Remarks:
Based on CLP and GHS
Conclusions:
Under the experimental conditions of this study, the test material (2-tert-butyl-1,4-dimethoxybenzene), was considered to be non-irritant to Reconstructed human Cornea-like Epithelium.

Based on the results of this study, the test material should not be classified for ocular irritation under GHS (2017) EU and Regulation (EC) No. 1272/2008.
Executive summary:

A Key in vitro study, was conducted to predict the acute eye irritation potential of the test material (2-tert-butyl-1,4-dimethoxybenzene) by measurement of its cytotoxic effect on the EpiOcularTM cornea epithelial model, in accordance with OECD Test Guideline No. 492 (25 June 2018).

 

Preliminary tests were performed to detect the ability of the test material to directly reduce MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) as well as its colouring potential. Following the preliminary tests, the eye irritation potential of the test material was assessed in the main test.

 

The test material and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 30 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature to remove any remaining test material from the tissue, blotted on absorbent material, and then incubated for another 2 hours at 37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

 

In the preliminary tests, the test material was neither found to have direct MTT reducing properties, nor colouring potential. In the main test, all acceptance criteria were fulfilled and the study was therefore considered to be valid. The relative mean viability of the tissues treated with the test item was 98% with a difference of 8% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.

 

Under the experimental conditions of this study, the test material (2-tert-butyl-1,4-dimethoxybenzene), was considered to be non-irritant to Reconstructed human Cornea-like Epithelium.

 

Based on the results of this study, the test material should not be classified for ocular irritation under GHS (2017) EU and Regulation (EC) No. 1272/2008.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2018 - 14 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
14 February 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test material: 2-tert-butyl-1,4-dimethoxybenzene
Public name: Vetimoss
EC number: 244-216-5
CAS number: 21112-37-8
Batch number: A180118C
Appearance: Clear, almost colourless liquid
Purity**: 99.78%
Expiry date: 06 June 2019
Storage conditions: Room temperature (15-25°C, ≤ 70% relative humidity (RH)), under inert gas, protected from humidity (tightly closed container)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Formulation: The test item was applied in its original form; no formulation was required
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
TISSUES (EYES)
- Source animal: Chicken
- Strain: ROSS 308
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129, Hungary)
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation: approximately 2.45 kg

Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption.

After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Citoxlab Hungary Ltd. and processed approximately within 2 hours of collection.
Vehicle:
physiological saline
Controls:
other: Positive Control: Benzalkonium chloride solution, 50% in water; Negative Control: Physiological saline (Salsol solution, 0.9% (w/v) NaCl)
Amount / concentration applied:
TEST MATERIAL
- 30 µL of the undiluted test item was applied onto the surface of the cornea attempting to cover the entire cornea surface uniformly with the test item

CONTROLS
- Positive control: 30 µL of 5% (w/v) Benzalkonium chloride solution
- Negative control: 30 µL of physiological saline (0.9% (w/v) NaCl) solution
Duration of treatment / exposure:
Exposure period of 10 seconds
Observation period (in vivo):
30, 75, 120, 180 and 240 minutes after the post-treatment rinse
Number of animals or in vitro replicates:
- Three test item treated eyes
- Three positive control treated eyes
- One negative control eye
Details on study design:
QUALITY CHECK OF THE ISOLATED CORNEAS
The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. The fluoresceintreated cornea was examined with a hand-held slit lamp with the eye in the head, to ensure that the cornea was not damaged. Only cornea in good condition was used for the study.

SELECTION AND PREPARATION OF CORNEAS
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head. A slight pressure was applied to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus.

The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

The selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected.
The cornea thickness was measured, any eye with cornea thickness deviating more than 10% from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced.
The selected eyes were acclimatized for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were maintained at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

NUMBER OF REPLICATES, NEGATIVE CONTROL USED and POSITIVE CONTROL USED
Test Item: three
Positive control: three
Negative control: one

APPLICATION DOSE AND EXPOSURE TIME
30 µL of the undiluted test item was applied onto the surface of the cornea attempting to cover the entire cornea surface uniformly with the test item. The exposure time was 10 seconds.

TREATMENT METHOD: Closed chamber except for manipulations and examinations, to maintain temperature and humidity.

REMOVAL OF TEST SUBSTANCE
The cornea surface was rinsed thoroughly with at least 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.


METHODS FOR MEASURED ENDPOINTS:
- Corneal thickness
- Corneal opacity
- Cornea swelling
- Measurements: A Haag-Streit Bern 900 slit-lamp microscope, with depth-measuring device no. 1 and slit-width setting at 9½.

SCORING SYSTEM DECISION CRITERIA
- See “background information box”
Irritation parameter:
percent corneal swelling
Run / experiment:
1 - up to 75%
Value:
0.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Run / experiment:
1 - up to 240 min
Value:
1.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
1
Value:
0.83
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
1
Value:
0.67
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Hystorical controls: yes - values within the ranges
Interpretation of results:
GHS criteria not met
Conclusions:
The test iteam Vetimoss (2-tert-butyl-1,4-dimethoxybenzene) CAS 21112-37-8 is not classified as a severe irritant and not classified as non-irritant, according to the results of this study.
Executive summary:

The aim of this study was to evaluate the eye irritation potential of the test material Vetimoss (2-tert-butyl-1,4-dimethoxybenzene), CAS number 21112-37-8. The test was performed using isolated chicken’s eyes in vitro eye irritation test. The study was conducted according to the OECD 438 Guideline and under GLP conditions.

 

The selected eyes were held in a horizontal position and the test item was applied onto the centre of the cornea in order to cover the surface of the cornea. The cornea was exposed to the test item for 10 seconds. Then the surface of the eyes was rinsed with physiological saline solution.

Three eyes were treated with 30 µL of the undiluted test item and three positive control eyes were treated in a similar way with 30 µL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution).

To evaluate the irritation potential, the corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects were observed and recorded. 

 

The negative control and positive control results were within the historical control data range in the experiment. The experiment was considered to be valid.

 

The result of the study showed no significant corneal swelling during the four-hour observation period post exposure. Slight cornea opacity change (severity 0.5 on one eye and severity 1.0 on two eyes) was observed in three eyes. Slight fluorescein retention change (severity 0.5 on two eyes and 1.0 on one eye) was recorded in all three eyes. However, no other corneal effects were observed.

 

Based on the result of this study, the test item Vetimoss (2-tert-butyl-1,4-dimethoxybenzene) is not classified as a severe irritant and not classified as non-irritant.

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

Based on the results observed in skin and eye irritation studies, the test material (2-tert-butyl-1,4-dimethoxybenzene) does not meet the criteria for classification as a skin or ocular irritant under GHS (2017) and EU Regulation (EC) No. 1272/2008.