(2-hydroxy-1,1-dimethylethyl)ammonium toluene-4-sulphonate

Endpoint summary

Description of key information

The test item propan-1 -ol,2 -amino-2 -methyl-, 4 -methylbenzenesulphonate was tested in a Corrositex study (OECD 435) for its corrosive properties (Eurofins, 2020) followed by an EpiDermTM study for its irritating properties (Eurofins, 2020). Based on the results of these studies the substance propan-1 -ol,2 -amino-2 -methyl-, 4 -methylbenzenesulphonate has to be classified as “irritant” in accordance with UN GHS “Category 2”. Based on results of an OECD 437 study (Bovine Corneal Opacity and Permeability, MB Research, 2015), the test item is considered to be a severe/corrosive eye irritant. Because the test item used in the OECD 437 study consisted of about 49,5 % Butan-1-ol, which is classified as a Category 1 eye irritant, a new study was conducted to examine the properties of the pure substance. Based on results of the OECD 496 study (Ocular Irritection, INT.E.G.RA, 2021), the test item is considered to be a severe/corrosive eye irritant in accordance with UN GHS “Category 1”.

 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-01-21 to 2020-03-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Version / remarks:

version 2015-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

artificial membrane barrier model

Remarks:

In vitro Membrane Test Corrositex™- Assay

Justification for test system used:

The CORROSITEX™ Assay is a standardized, quantitative in vitro test for skin corrosivity and has been validated by the ECVAM for testing acids, bases and their derivatives [11]. The bio-barrier membrane is constructed to have physico-chemical properties similar to rat skin.

Details on test system:

CORROSITEX™ Assay Kit (Invitro International; Irvine, CA 92614, Lot No.: CT040119

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

100 mg of the test substance were addes to Tube A & B
500 mg of the test item were applied on top of the BIOBARRIER

Duration of treatment / exposure:

Table 1: Packing Group Designation
Category Time [min.] Time [min.] Time [min.] Time [min.]
1 0 – 3 > 3 - 60 > 60 - 240 > 240
2 0 - 3 > 3 - 30 > 30 - 60 > 60

Number of replicates:

4x BIOBARRIER discs

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The corresponding BIOBARRIER has been treated with
- negative control: 500 µl of 10% Citric acid (in ddH2O)
- positive control: 500 µl of 85% Phosphoric Acid
- 500 g of solid test item

Duration of treatment / exposure:

based on the confirm experiment in the CORROSITX™, the chemical has been categorized in category 2. Therefore, the CDS solution was observed for 1 h. B

Observation period:

1 h

Details on study design:

BIOBARRIER Prepartion:
-The content of the BIOBARRIER diluent was added to the vial of BIOBARRIER matrix powder.
-The vial was heated to 68°C (± 1°C) in a water bath under smooth agitation.
-After complete dissolution the solution was allowed to sit for 5 min.
-200 µL of the BIOBARRIER were pipetted into each membrane disc.
-The BIOBARRIERS were set on the tray and kept in the cold (2 - 8°C) for at least 2 h before the test was conducted

Categorization Test:
- 100 mg of the test substance were added to the tubes A and B.
- After shaking a colour change was observed in either of the tubes and colour was matched to the corresponding colour charts on the CORROSITEX™ Testing Protocol Poster.
- If no colour change had been observed in either tube, CONFIRM reagent was added to Tube B. After shaking, the resulting colour was matched to the colour chart on the CORROSITEX™ Testing Protocol Poster.
If the test item has a strong inherent colour or shows other characteristics impairing a clear categorization according to the colour chart, the pH value can be measured in tubes A and B and is used to confirm/determine the category of the test item, according to the Corrositex® Reference Manual .
 
Classification Test:
- The CDS vials were warmed to room temperature
- Vials 1 - 4: sample replicate testing
- Vial labelled (+): positive control sample,
-Vial labelled (-) :negative control.
- Vial labelled C: CDS colour control.
- 1x BIOBARRIER disc was added on top of the first vial
- 500 mg of the test item were applied evenly on the top of the BIOBARRIER disc and starting time was recorded, this was repeated for the remaining vials, staggering each start time by e.g. 10 seconds.
The start time difference for each vial was subtracted from the final time to determine the net response time.
As soon as a reaction had been observed, the time was recorded.

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

Corrositex™

Value:

> 60

Negative controls validity:

valid

Remarks:

n.a. for 10% Citrix Acid

Positive controls validity:

valid

Remarks:

26.08 min for 85% Phosphoric Acid

Table2:  Results of the Test Item Propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate

	CORROSITEX™ Time [min]	Colour Change
Replicate 1	n.a.	no
Replicate 2	n.a.	no
Replicate 3	n.a.	no
Replicate 4	n.a.	no
Mean ± SD	n.a.
Positive control	26.08	yes
Negative control	n.a.	no

During the observation period of 1 h the CDS was not activated.

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not corrosive to skin.

Executive summary:

The potential of the test substance to induce skin corrosion was analysed by using the “In VitroMembrane Barrier Test (CORROSITEX™ Assay)”, comprising a Chemical Detection System covered by a bio-barrier membrane.

The test substance Propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate proved its ability to activate the CDS and was subsequently subjected to the timescale category test. The test substance was applied undiluted on top the bio-barrier membraneand the timerequired to activate the CDSwas measured in four replicates. The mean time was compared to the given thresholds (see Table 1).

The test substance was compatible with the CORROSITEX™ Assay, as assessed in the qualification step. The categorization step and the classification step could be performed.

A direct colour change was not observed. CONFIRM reagent was added to tube B and the category was read from the CORROSITEX™ colour chart. The chemical has been categorized to category 2.

In this study under the given conditions the test substance showed no corrosive effects as the mean time, required to activate the CDS, was > 60 min. (category 2). 

The controls confirmed the validity of the study. The positive control activated the CDS between 3 - 60 min. (26.08 min.), the negative control did not activate the CDS before 60 min.

 

This in vitro study was performed to assess the corrosive potential of test item by means of the in vitro Membrane Barrier Test for

Skin Corrosion (OECD TG 435) using the Corrositex™ test kit.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-03-19 to 2020-06-08

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)

Version / remarks:

version 2019-06-18

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Version: 2019-06-31

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiDermTM

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS “Category 2”. Depending on the regulatory framework it can also be used to identify non-classified chemicals

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDerm tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm2); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 30854)
2x 24-well plates
8x 6-well plates
1x bottle of assay medium (DMEM-based medium, Lot No.: 032620MJC)
1x bottle of DPBS Rinse Solution (Lot No.: 012120MJA)
1x 1 vial 5% SDS Solution (TC-SDS-5%) (Lot No.: 110519MSA)
25 pieces Nylon Mesh circles (8 mm diameter, 200 μm pore)

Further Reagents
MTT solution
- MTT stock solution: 5 mg/mL MTT (VWR; Lot 18I1156332) in PBS (Gibco; Lot No.: 2172337)
- MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL)
Isopropanol (AppliChem; Lot No.: 0001826136)
Dist. Water (Sigma; Lot No.: RNBH8991)

Pre-Experiments
To check the non-specific MTT-reducing capability of the test item 25 mg of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator.
The mixture did not turn blue/purple. Thus, the additional test with freeze-killed tissues and the quantitative corrections were not necessary.
To check the colouring potential of the test item 25 mg of the test item were mixed per 300 μL aqua dest. and per 300 μL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min. The mixture of 25 mg of the test item per 300 μl aqua dest. showed colouring detectable by unaided eye-assessment. The mixture with isopropanol did not show any colouring. Therefore, the absorption of the chemical in water was measured in the range of 570 ± 30 nm. No absorption was measured in the relevant range. Therefore, NSCliving equalled 0%.
The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

Experimental Procedure
Upon receipt of the EpiDermTM, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL assay medium per well. The surface was dried using a sterile cotton tip and the plates were incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 for 60 ± 5 min. Subsequently the tissues were transferred into new wells containing 0.9 mL pre-warmed assay medium per well and were incubated for 18 ± 3 h in a humidified incubator at 37 ± 1 °C, 5.0% CO2.
After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue and occurred sequentially for the other tissues, e.g. in one-minute intervals. After dosing of all tissues, all plates were incubated for 25 ± 1 min under the sterile flow and for the remaining time of 35 ± 1 min transferred to the incubator. Then the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper. The inserts were placed in prepared new 6-well plates containing 0.9 mL pre-warmed fresh assay medium per well and the tissue surface was dried using a sterile cotton tip. The plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h.
After this post-incubation period the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a prepared 24-well plate containing 300 μL pre-warmed MTT medium. This plate was incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%.
After the MTT incubation period, the tissues were rinsed three times with DPBS and afterwards placed on blotting paper to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature for at least 2 h with gentle shaking on a plate shaker.
Before using the extracts, the plate had been shaken for at least 15 min on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 μL aliquots per each tissue were transferred into a 96-well plate. OD was measured at 570 nm with a filter band pass of maximum ± 30 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 25 μL DPBS


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL DPBS
- Concentration (if solution):

POSITIVE CONTROL
- Concentration (if solution): 30 μL 5% SDS solution

Duration of treatment / exposure:

After dosing of all tissues, all plates were incubated for 25 ± 1 min under the sterile flow and for the remaining time of 35 ± 1 min transferred to the incubator.

Duration of post-treatment incubation (if applicable):

The plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h.
After this post-incubation period the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a prepared 24-well plate containing 300 μL pre-warmed MTT medium. This plate was incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%.

Number of replicates:

The test was performed on a total of 3 tissues per dose group.

Species:

other: in vitro

Vehicle:

unchanged (no vehicle)

Irritation / corrosion parameter:

% tissue viability

Remarks:

test meets acceptance criteria : standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.

Value:

14.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

mean absolute OD570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8

Positive controls validity:

valid

Remarks:

mean relative tissue viability of the three positive control tissues is ≤ 20%

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.830). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (4.2%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.6% - 14.9%).

Pre-Experiments

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 25 mg of the test item per 300 μl aqua dest. showed colouring detectable by unaided eye-assessment. The mixture with isopropanol did not show any colouring. Therefore, the absorption of the chemical in water was measured in the range of 570 ± 30 nm. No absorption was measured in the relevant range. Therefore, NSCliving equalled 0%.

The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

Conclusions:

In this study under the given conditions the test item showed irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was ≤ 50% (14.4%).

Executive summary:

In the present study the skin irritant potential of Propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404, [7]) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 25 mg of the test item per 300 μl aqua dest. showed colouring detectable by unaided eye-assessment. The mixture with isopropanol did not show any colouring. Therefore, the absorption of the chemical in water was measured in the range of 570 ± 30 nm. No absorption was measured in the relevant range. Therefore, NSCliving equalled 0%.

The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (14.4%) after 60 min treatment and 42 h post-incubation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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

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.

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.

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

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

 

RESULTS OF TEST ITEM

Dose	Sample OD 405	Net OD 405	Irritancy Score
25 mg	0,500	0,500	22,2*
50 mg	0,625	0,626	27,8**
75 mg	0,441	0,439	19,5
100 mg	0,723	0,710	30,7 a
125 mg	0,679	0,675	30,0

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

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

Dose	Sample OD 405	Net OD 405	Irritancy Score
25 mg	0,500	0,500	22,2*
50 mg	0,625	0,626	27,8**
75 mg	0,441	0,439	19,5
100 mg	0,723	0,710	30,7 a
125 mg	0,679	0,675	30,0

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

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The bovine eyes, in a Hanks’ Balanced Salt Solution (HBSS) with penicillin-streptomycin, were received from Spear Products on 29 Oct 2015 and transported to MB Research in a refrigerated container.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Three bovine corneas per group were dosed with 0.75 ml of Catalyst 1786B,
Batch# 1776194, Minimal Essential Media (MEM) (negative control), or 100% Ethanol (positive control). Following a 10-minute exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined.

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

See Details on study design

Number of animals or in vitro replicates:

Not applicable

Details on study design:

The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded.
Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS.
The dissected corneas were mounted in specially designed holders that were separated into anterior and posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects.
The entire holder was incubated at 32ºC (± 1º) and allowed to equilibrate for at least one hour but not longer than two hours.
A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied with the opacitometer. Any cornea with a value greater than 7 units was discarded.
Following the pretest observations, the MEM solution was removed from the anterior chamber. A volume of 0.75 ml of ethanol, MEM or test article was applied to the epithelium of each of the three positive controls, three negative controls or three test article-treated corneas in a manner, which ensured the entire cornea was covered. The closed-chamber method of dose administration was used.
All holders and corneas were placed in a horizontal position (anterior side up) in the 32ºC (± 1º) incubator. After 10 (± 1) minutes, the test article, ethanol or MEM solution was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.
All corneas were incubated at 32ºC (± 1º) for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the
OP-KIT. This reading was used in the final IVIS calculations.
Immediately following the two-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium fluorescein solution in Dulbecco's Phosphate Buffered Saline (PBS). Each holder was returned to the 32ºC (±1º) incubator in a horizontal position (anterior side up) ensuring contact of the fluorescein with the cornea.
After 90 (± 5) minutes, the fluid from the posterior chamber was removed and the amount of dye, which passed through the cornea (permeability), was measured as the optical density at 490 nm by a spectrophotometer. A 1:1000 dilution of the fluorescein was prepared and measured in the spectrophotometer as a measure of consistency.
Data analysis
Individual corrected opacity scores were calculated by subtracting the pretest score from the ten-minute and two-hour scores. Corrected mean opacity scores were calculated by averaging the individual two-hour corrected opacity scores for a given dose group and subtracting the mean opacity score for the negative control group. A corrected mean opacity score was not calculated for the negative control, rather only the mean of the individual two-hour corrected opacity scores were calculated (with no subtraction of mean opacity score for negative control.)
Individual corrected optical densities were calculated by subtracting the mean optical density for the negative control group from the individual optical density values. Individual corrected optical densities were not calculated for the negative control group. Corrected mean optical densities were calculated by averaging the individual corrected optical density values for a given dose group. A corrected mean optical density was not calculated for the negative control, rather only the mean of the individual optical densities was calculated.
The In Vitro Irritancy Score (IVIS) for the test article and Vehicle control was calculated by adding the corrected mean opacity score to fifteen times the corrected mean optical density, as shown by the equation below. The calculation to obtain an IVIS for the positive control was performed in the same manner as the test article.
In Vitro Irritancy Score (IVIS) = Corrected Mean Opacity Score + (15 x Corrected Mean Optical Density Score)
The calculation to obtain an IVIS for the negative control was performed by adding the mean opacity score to fifteen times the mean optical density, as shown by the equation below.
In Vitro Irritancy Score (IVIS) = Mean Opacity Score + (15 x Mean Optical Density Score)

Based on the IVIS score, the test article was classified according to the prediction model described in DB-ALM Protocol No. 1271, a modification of the prediction model suggested by Gautheron, et al. (1994). Results from test situations should be compared to known materials tested under similar conditions. This classification system is the most consistent with recent regulatory reviews of the assay performance.
IVIS CLASSIFICATION
0 to 3 Non-Irritant
3.1 to 25 Mild Eye Irritant
25.1 to 55 Moderate Eye Irritant
55.1 and above Severe/Corrosive Eye Irritant
OECD Guideline #437 defines a substance that produces an IVIS of > 55 as Category 1, a substance that causes “Serious eye damage.”
IVIS UN GHS2
≤ 3 No Category
>3 to ≤55 No prediction can be made
>55 Category 1
The assay was accepted because the positive control had an IVIS that fell between two standard deviations of the historical mean.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean value for 3 experiments

Value:

74.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Mean of 3 experiments

Value:

61

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on an In Vitro Irritation Score of greater than 55, and as defined in OECD Guideline 437, the test article is in UN GHS Category 1. According to EURL ECVAM DB-ALM Protocol No. 127, Catalyst 1786B, is considered to be a severe/corrosive eye irritant.
Based on this result, also propan-1-ol,2-amino-2-methyl-, 4-methylbenzenesulphonate is considered severe/corrosive eye irritant.

Executive summary:

Based on results of OECD 437 study (Bovine Corneal Opacity and Permeability) the test item is considered to be a severe/corrosive eye irritant. Based on this result, also propan-1-ol,2-amino-2-methyl-, 4-methylbenzenesulphonate is considered severe/corrosive eye irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Additional information

Based on results of OECD 437 study (Bovine Corneal Opacity and Permeability)study, Catalyst 1786B is considered to be a severe/corrosive eye irritant. Based on this result, also propan-1-ol,2-amino-2-methyl-, 4-methylbenzenesulphonate is considered to be a severe/corrosive eye irritant.

Justification for classification or non-classification

Based on the results of the in vitro skin and eye irritation studies and according to the criteria of EC Regulation 1272/2008 and subsequent regulations on classification, labelling and packaging of substances and mixtures, propan-1-ol,2-amino-2-methyl-, 4-methylbenzenesulphonate has to be classified into category 2 for skin irritation (H 315) as well as into category 1 for eye damage (H319).