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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:
20.07.2021 to 15.12.2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 496 (In vitro Macromolecular Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2019
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(2-hydroxy-1,1-dimethylethyl)ammonium toluene-4-sulphonate
EC Number:
269-537-8
EC Name:
(2-hydroxy-1,1-dimethylethyl)ammonium toluene-4-sulphonate
Cas Number:
68298-05-5
Molecular formula:
C7H8O3S.C4H11NO
IUPAC Name:
(2-hydroxy-1,1-dimethylethyl)ammonium toluene-4-sulphonate
Test material form:
solid
Details on test material:
Propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate from Evonik Batch NC19408606

Test system

Controls:
yes
Amount / concentration applied:
- test item: 25, 50, 75, 100, 125 mg .
- 125 μl of quality control QC1 and QC2, and the four calibrator CAL0, Cal1, Cal2 and CAL3
Duration of treatment / exposure:
The macromolecular matrix of the Ocular Irritection® test method is exposed to the
test chemicals and concurrent controls for 24.0 ± 0.5 hours in an incubator maintained at
25 ± 1°C.
Number of animals or in vitro replicates:
2
Details on study design:
DAY1
DETERMINATION OF SAMPLE PH:
The pH values was determined by measuring the 10% dilution ( v/v or w/v) of test substance; applicability domain range: > 4 and < 9

DETERMINATION OF SAMPLE PROPERTY: SURFACTANT OR NON-SURFACTANT
A 10% solution of the sample was tested with foam testing to be performed to determine whether the compound should be tested utilizing the surfactant or non surfactant application procedure.

REAGENT PREPARATION
As a basis of the Ocular Irritection® in vitro macromolecular test method, a macromolecular matrix is prepared by dissolving the reagent powder provided within the kit into a hydrating solution, and filtering the dissolved reagent. The resulting pH was measured: 8.032 and it has been included in the range 7.91 – 8.19 of specification data sheet . Furthermore, the reagent solution (as well as the blanking buffer conducted in parallel for each tested dose/concentration) was activated using an activator buffered solution, to reduce the pH of the reagent solution and initiate formation of the ordered macromolecular matrix. The resulting pH of the activated reagent solution was measured: 6.612 and it has been included in the range 6.42 – 6.74 of specification data sheet. Aliquots of the activated protein matrix reagent solution are transferred to a 24-well plate.

TEST ITEM EXPOSURE PROCEDURE
The Reagent solution and the Ocular Blanking activated buffer were dispensed in the wells of 24-well plate supplied with the kit, in an established layout based on the number of wells required to analyze the four calibrators, the 2 quality controls and 5 doses of the test item. The 5 weights of the test substance was dispensed onto Membrane Discs and insert them into the corresponding wells with contained the reagent solution and the blanking buffer. 125 μl of quality control QC1 and QC2, and the four calibrator CAL0, Cal1, Cal2 and CAL3 are applied neat onto the membrane disc placed over the matrix reagent

INCUBATION
The macromolecular matrix of the Ocular Irritection® test method is exposed to the test chemicals and concurrent controls for 24.0 ± 0.5 hours in an incubator maintained at 25 ± 1°C.

DAY2
REMOVING MENBRANE DISCS
After 24 h of incubation the plates were removed from the thermostat. The lids were removed from the assay plates. Secondly the plastic wrap was removed from each assay plate as well. Thirdly each membrane disc was taken out individually with plastic forceps.
The membranes were checked for any damage and it has been verified that all membrane discs were intact. If a membrane damage had occurred, it should have been recorded. A wooden stirring stick was used to scrape the Reagent and Blank wells to ensure that all the precipitate was lifted from the bottom of each well. One stirring stick was used for each sample, starting from the lowest dose wells first, progressing to wells higher dose, and a separate stirring stick for each calibrator well.
There wasn’t any reduction of volume in the wells. Reduced volumes may be indicative of hygroscopic effects or technical problems, and it should be recorded. For this assay all membrane discs were intact, and no volume reduction was noticed.

TRANSFERRING FROM 24 WEELS ASSAY TO A 96 WELL READING PLATE
Test chemicals and controls are transferred to a 96 well plate for OD reading at 405nm. Each 24-well was transfered to single 96-well, of the 96 well plate. The raw OD readings from each well are obtained and the IDE scores for the QCs and test chemicals are calculated by the Irritection®software. MQS for a test chemical is determined from a single test run qualified as appropriate based on the analysis of the OD scores for the calibrators and QC chemicals as well as aspects of the dose response generated with the five tested doses/concentrations of test chemical.

DATA RECORDING
The data of optical density measured by the spectrophotometer Tecan Sunrise Basic for OD reading at 405nm, are recorded automatically by Irritection®software.

INTERPRETATION OF RESULTS AND PREDICTION MODEL
The Irritection Software program serves as the user interface to plate reader. The program automatically receive the optical density reading from plate reader and then convert the data to the Irritection Draize Equivalent (IDE) score. The optical density (OD405) obtained with a qualified test chemical is compared to the standard curve obtained with the set of calibrators, to derive an Irritection Draize Equivalent (IDE) Score, for each tested dose/concentration. The highest obtained IDE score, named the Maximal Qualified Score (MQS), is then used to predict the ocular hazard potential of the test chemical according to the UN GHS classification system. In the case of the Ocular Irritection® in vitro macromolecular test method the following Prediction Model is used:
Maximal Qualified Score (MQS)/ Predicted UN GHS classification
0.0 – 12.5/ No Category
>12.5 – 30/ No Prediction can be made
>30/ Category 1

ACCEPTANCE CRITERIA OF THE STUDY
All data are calculated and analyzed via a computer Irritection®software program which determines assay result acceptance based upon qualification parameters defined in the program (Irritection Software). In general, the Irritection Software has been designed to accept sample data as qualified if the following criteria are met. One of two criteria relating to four calibrators and two Quality Controls must be met for a test run to be accepted as Qualified for further data analysis:
‒ The values obtained for all four calibrators and for at least one of two Quality Controls are within the pre-established accepted ranges; or
‒ The values obtained for any three of four calibrators, and for both Quality Controls are within the pre-established accepted ranges. If only one calibrator is out of its acceptance range, the OI® software substitutes a pre-defined value for generation of the standard curve
An OI® test run is considered Non-Qualified (NQ) when either two (or more) calibrators are out of range, or when one calibrator and one Quality Control are out of range.
- Acceptance OD405 range:
Calibrator 0: 0.062 - 0.262
Calibrator 1: 0.089 - 0.315
Calibrator 2: 0.351 - 0.945
Calibrator 3: 1.277 - 2.127
- Acceptance IDE range:
QC 1: 7.2 - 20.8
QC 2: 23.6 - 35.6
Acceptance criteria for calibrators and quality control chemicals in the Ocular Irritection® test method:
- Sample blanks are less than 500 OD units;
- the net sample OD is greater than -15
- If the Net ODχ for a test chemical in a qualified run is below OD Cal 2, an additional check is prompted to verify that the macromolecular matrix is responding properly. This check is performed by addition of an inhibition check solution provided in the test kit followed by re-measuring the ODχ which should fall above ODCal 2 for the data to qualify/be accepted for further interpretation
- A dose response check is conducted to verify that the test chemical dose response is consistent with a typical pattern characteristic for known types of correctly predicted chemicals, If the dose response for a test chemical has an atypical/irregular pattern, the IDE results should be excluded from consideration for MQS determination.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
max. irritancy score
Run / experiment:
100 mg substance
Value:
30.7
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Calibrators and QC substances in the acceptance range:
Designation OD 405 values Irritancy Score Range Limit (OD)
Cal0 0,166 0,0 0,062 – 0,262
Cal1 0,284 12,5 0,089 – 0,315
Cal2 0,674 30,0 0,351 – 0,945
Cal3 1,749 51.0 1,277 – 2,127

Designation OD 405 values Irritancy Score Range Limit (Score)
QC1 0,347 15,3 7,2 - 20,8
QC2 0,804 32,5 23,6 - 35,6

The DETERMINATION OF SAMPLE pH was conducted by measuring the 10% dilution ( v/v or w/v) of test substance. The pH was 6.849 and it has been included in the applicability domain range of > 4 and < 9. The sample resulted suitable for Ocular Irritection®.

The DETERMINATION OF SAMPLE PROPERTY (SURFACTANT OR NON-SURFACTANT) was conducted to determine whether the compound should be tested utilizing the surfactant or non surfactant application procedure. As the sample resulted as NON – SURFACTANT it was utilized the non surfactant application procedure for this study.

During the Day 2 the membranes were checked for damage. For this assay all membrane discs were intact, and no wells were detected as volume reduction.

The irritancy of the test sample is judged to be defined by highest qualified score calculated by the Irritection software. Maximum Qualified Score (MQS 30,7) is selected from data because it is the irritancy score that correlates most closely with the in vivo irritancy properties of test item.

Any other information on results incl. tables

pH:


The pH values was determined by measuring the 10% dilution ( v/v or w/v) of test substance. The pH was 6.849 and it has been included in the applicability domain range of > 4 and < 9 The sample is suitable for Ocular Irritection®.


 


DETERMINATION OF SAMPLE PROPERTY: SURFACTANT OR NON-SURFACTANT
The sample resulted as NON – SURFACTANT it was utilized the non surfactant application procedure for this study.


 


RESULTS OF CONTROL ITEMS



































DesignationOD 405 valuesIrritancy ScoreRange Limit (OD)
Cal00,1660,00,062 – 0,262
Cal10,28412,50,089 – 0,315
Cal20,67430,00,351 – 0,945
Cal31,74951.01,277 – 2,127

 























DesignationOD 405 valuesIrritancy ScoreRange Limit (Score)
QC10,34715,37,2 - 20,8
QC20,80432,523,6 - 35,6

 


RESULTS OF TEST ITEM









































DoseSample OD 405Net OD 405Irritancy Score
25 mg0,5000,50022,2*
50 mg0,6250,62627,8**
75 mg0,4410,43919,5
100 mg0,7230,71030,7 a
125 mg0,6790,67530,0

a Maximum Qualified Score
* examine dose curve
**dose eliminated

Applicant's summary and conclusion

Conclusions:
The test item was evaluated with OCULAR IRRITECTION (OI®) test method (OECD 496). The following weights of sample were applied for analysis: 25, 50, 75, 100, 125 mg. The results demonstrated that the sample can be considered a Category 1 with a Maximum Qualified Score of 30.7.
Executive summary:

The test item was evaluated with OCULAR IRRITECTION (OI®) test method (OECD 496). The following weights of sample were applied for analysis: 25, 50, 75, 100, 125 mg. The results demonstrated that the sample can be considered a Category 1 with a Maximum Qualified Score of 30.7.


 


The DETERMINATION OF SAMPLE pH was conducted by measuring the 10% dilution ( v/v or w/v) of test substance. The pH was 6.849 and it has been included in the applicability domain range of > 4 and < 9. The sample resulted suitable for Ocular Irritection®.


 


The DETERMINATION OF SAMPLE PROPERTY (SURFACTANT OR NON-SURFACTANT) was conducted to determine whether the compound should be tested utilizing the surfactant or non surfactant application procedure. As the sample resulted as NON – SURFACTANT it was utilized the non surfactant application procedure for this study.


 


Calibrators and QC substances in the acceptance range.


 


During the Day 2 the membranes were checked for damage. For this assay all membrane discs were intact, and no wells were detected as volume reduction.


 


The irritancy of the test sample is judged to be defined by highest qualified score calculated by the Irritection software. Maximum Qualified Score (MQS 30,7) is selected from data because it is the irritancy score that correlates most closely with the in vivo irritancy properties of test item.


 


RESULTS OF TEST ITEM









































DoseSample OD 405Net OD 405Irritancy Score
25 mg0,5000,50022,2*
50 mg0,6250,62627,8**
75 mg0,4410,43919,5
100 mg0,7230,71030,7 a
125 mg0,6790,67530,0

a Maximum Qualified Score
* examine dose curve
**dose eliminated