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Diss Factsheets

Toxicological information

Eye irritation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-tert-butyl-1,4-dimethoxybenzene
EC Number:
244-216-5
EC Name:
2-tert-butyl-1,4-dimethoxybenzene
Cas Number:
21112-37-8
Molecular formula:
C12H18O2
IUPAC Name:
2-tert-butyl-1,4-dimethoxybenzene
Test material form:
liquid
Details on test material:
Name: 2-tert-butyl-1,4-dimethoxybenzene
Public name: Vetimoss
EINECS No.: 244-216-5
CAS No.: 21112-37-8
Batch No.: A180118C
Description: Colourless to pale yellow liquid
Molecular weight: 194.27 g/mol
Purity: 99.78%
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.

Test animals / tissue source

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.

Test system

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%.

Results and discussion

In vitro

Results
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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.