Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1)

Endpoint summary

Description of key information

The in vitro skin corrosion of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 431. The Relative mean tissue viability calculated as a percentage of the negative control was 47.6% after the 60 minutes exposure and 76% after the 3 minutes exposure. The test item did not meet the criteria for classification as corrosive to the skin according to the criteria laid down in the OECD Guideline for Testing of Chemicals 431.

 

The in vitro skin irritation of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 439. The percentage of viability obtained with the test item Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) was 34.233%, therefore it has to be considered as irritant to the skin according to the criteria laid down in the OECD Guideline for Testing of Chemicals 439. All the acceptance criteria were met and the study is therefore considered as valid.

 

The in vitro skin irritation of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 437. An In Vitro Irritation Score (IVIS) of 20.69 was calculated for Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) from corneal opacity and permeability measurements. No prediction can be made if the IVIS is >3 but ≤ 55 and, therefore no conclusion can be made based on the criteria laid down in the OECD Guideline for Testing of Chemicals 437. All the acceptance criteria were met and the study is therefore considered as valid.

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:

From March 27, 2017 to April 06, 2017

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)

Version / remarks:

Updated Guideline adopted July 29, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: Human Skin Model Test with EpiDermTM tissues models

Cell type:

other: Normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis

Justification for test system used:

The In vitro Skin Corrosion: Human Skin Model Test is based on the observation that skin corrosion (necrotic damage of viable skin cells) shows a high correlation with skin cell cytotoxicity, occurring rapidly after brief exposure of the skin barrier (stratum corneum) to a corrosive chemical. It is designed to predict and classify the skin corrosivity potential of a chemical by using a three-dimensional human epidermis model. Ultrastructurally, the skin models closely parallel human skin

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL TEST WITH EPIDERMTM TISSUES MODEL
- Model used: Human epidermis model derived from human keratinocytes and consists of normal, human-derived epidermal keratinocytes (NHEK)
- Date of initiation of testing: March 27, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Pre-incubation period: EpiDermTM tissues were pre-incubated at least 1 - 24 hours before dosing. Inserts were transferred from the refrigerator into 6-well plates containing pre-warmed assay medium (temperature not specified)
- Temperature used during treatment / exposure: EpiDermTM tissues were treated with the test item and a positive and negative control and prepared in duplicate. During treatment, EpiDermTM tissues within a set of well plates were kept at room temperature for a 3 ± 0.5 minute exposure period. The second replicate of well plates underwent a 60 ± 5 minute exposure period in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2)
- Temperature of post-treatment incubation: As part of the MTT-100 assay (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide), tissues were incubated for 3 hours (37 ± 1.5 °C, 5 ± 0.5 % CO2)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 50 µL (79.4 µL/cm2 according to guideline) of the test item and controls was dispensed directly onto duplicate EpiDermTM tissue surfaces. After the exposure periods, the tissues were removed from the 6-well plate and gently
rinsed using a wash bottle / multi-pipette containing DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: Yes, at the end of the exposure period the tissues were rinsed using DPBS instead of PBS specified in the guideline

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: The MTT-solution was prepared freshly on day of use (resulting: 1 mg/mL). MTT concentrate from MatTek, MTT diluent from MatTek
- Incubation time: Well plates containing MTT solution (300 µL) were prepared in the pre-warming period and kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required. Following rinsing after the exposure period, the tissues were transferred from the holding plates to the MTT-plates and incubated for a 3 hour period (37 ± 1.5 °C, 5 ± 0.5% CO2)
- Spectrophotometer: Microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1))
- Wavelength: 570 nm (OD570)
- Filter: No reference filter

NUMBER OF REPLICATE TISSUES: Tissues prepared in duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Justification: A test item may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT
- Pre-test for colour interference: 50 µL of the test item were added to 0.3 ml of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If the solution changed colour significantly, the test item is presumed to have the potential to stain the tissue
- Pre-test for direct MTT reduction: To test if an item directly reduces MTT, 50 µL of the test item were added to 1 ml of a MTT/DMEM solution (1 mg/mL) and were incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. If the MTT/DMEM solution (1 mg/mL) turns blue/purple, the test item reduces MTT and an additional test on freeze-killed tissues must be performed

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is < 50%, or if the viability after 3 minutes exposure is ≥50 % and the viability after 1 hour exposure is <15%
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is ≥50% and the viability after 1 hour exposure is ≥15%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL (79.4 µL/cm2 according to guideline) of the test item were dispensed directly onto duplicate EpiDermTM tissue surfaces

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL was applied to each set of duplicate tissues for the 3 min and 1 hour exposure periods

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL was applied to each set of duplicate tissues for the 3 min and 1 hour exposure periods

Duration of treatment / exposure:

Two exposure groups consisting of 3 ± 0.5 and 60 ± 5 minutes. After the pre-incubation was completed, the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2)

Duration of post-treatment incubation (if applicable):

Following the exposure period, tissues were rinsed and transferred from the holding plates to MMT-plates and incubated for 3 hours (37 ± 1.5 °C, 5 ± 0.5% CO2)

Number of replicates:

Duplicate

Type of coverage:

not specified

Preparation of test site:

other: At least 1 hour or maximum 24 hours before dosing, EpiDerm™ tissues were removed from the refrigerator to 6-well plates containing the pre-warmed assay medium

Vehicle:

unchanged (no vehicle)

Controls:

yes

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative absorbance (% of negative control)

Run / experiment:

3 minutes

Value:

76

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative absorbance (% of negative control)

Run / experiment:

60 minutes

Value:

47.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Optical evaluation after 1 hour revealed no blue colour and, therefore, the test item did not reduce MTT
- Colour interference with MTT: No colour interference noted

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The quality certificate of the supplier of the test kit demonstrating its robustness (treatment with 1% Triton X-100: 4.77 hours ≤ ET50 ≤ 8.72 hours) is annexed to the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, range: 1.557 - 1.681
- Acceptance criteria met for positive control: Yes, 3.4 %
- Acceptance criteria met for variability between replicate measurements: Yes, range: 1.2 - 13.7 %

Results after treatment with the reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) and the controls:

Dose Group	Exposure Interval	Mean Absorbance (OD) of 2 Tissues	CV (%)	Relative Absorbance (% of Negative Controls)
Negative Control	3 minutes	1.553	2.1	100.0
Positive Control		0.349	1.2	22.5
Test Item		1.179	2.6	76.0
Negative Control	60 minutes	1.583	5.6	100.0
Positive Control		0.054	0.2	3.4
Test Item		0.754	13.7	47.6

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean tissue viability calculated as a percentage of the negative control was 47.6% after the 60 minutes exposure and 76% after the 3 minutes exposure. In accordance with CLP Regulation (EC) No. 1272/2008, the substance can be considered as non-corrosive to the skin.

Executive summary:

An in vitro skin corrosion study was undertaken using a Human Skin Model Test with EpiDerm™ tissues model to determine the corrosive potential of the reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) in accordance with the OECD Testing Guideline 431. The test was GLP-compliant.

Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) did not reduce MTT ( 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide), neither did it dye water in a pre-test, indicating that the substance would not interfere with the MTT endpoint.

Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water), or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. The validity of the test system and the specific batch of the tissue models was confirmed according to the assay acceptability criteria. After exposure of the tissues to the test item the relative absorbance value decreased to 76.0% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 47.6%. Both values did not affect the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) is non corrosive to skin according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 17, 2017 to October 11, 2017

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:

July 28, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

Reconstructed human epidermis (EpiDerm™)

Source species:

human

Cell type:

other: Normal human-derived epidermal keratinocytes

Source strain:

other: Keratinocyte strain 00267

Justification for test system used:

Initially the predictive capacity of the modified EpiDerm Skin Irritation Test (SIT) test method, using MatTek EpiDerm™ tissue model EPI-200, underwent full prospective validation from 2003-2007. The test method components of this method were used to define the essential test method components of the original and updated ECVAM Performance Standards (PS). A modification of the original EpiDerm™ SIT was validated using the original ECVAM PS in 2008. In 2008, ECVAM Scientific Advisory Committee (ESAC) concluded that the Modified EpiDerm™ SIT has sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non-skin irritating) test substances.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed human epidermal model (EPI-200-MatTek Corporation). The EpiDerm™ tissue model is a multi-layered highly differentiated model of the human epidermis, consisting of organised basal, spinous and granular layers, and a multi-layered stratum corneum that contains intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo
- Lot number: 25842
- Date of receipt: September 19, 2017
- Supplier: MatTek Laboratories
- Storage: On the day of receipt (day 0), EpiDerm™ tissues were pre-incubated overnight at 37 °C, 5 % CO2, ≥95 % RH

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Application of test, positive control, and negative control material for 60 ± 1 minutes (25* minutes at room temperature and 35 minutes at 37 °C, 5 % CO2, ≥95 % RH). *Delays during the study meant that total incubation time was extended by 4 minutes, resulting in an increased incubation time at room temperature. The Study Director examined the positive and negative controls and concluded that these had passed the acceptance criteria for the assay, suggesting that no negative impact had occurred from the additional incubation
- Temperature of post-treatment incubation: 42 ± 4 hours prior to the MTT endpoint

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Exposure of test and reference items followed by rinsing steps, prior to the 42 ± 4 hour post-dose incubation

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE (as per SOP L0029)
- Wavelength: 570 nm (OD570)
- Filter: Without reference filter
- Spectrophotometer: BMG LabTech FluoStar Optima

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Tissue viability: 1.39 ± 0.046 (mean and SD of MTT value of 3 tissues exposed to H2O). Acceptance range is 1.0 - 3.0
- Skin barrier function (ET50 value for 1 % Triton X-100) where ET50 is the time taken for 1 % Triton X-100 to reduce the viability of the skin model to 50 % relative to the negative control: 7.12 hours, within the acceptance range of 4.77 - 8.72 hours.
- Morphology: Tissue thickness of 95.5 μm. Acceptance range is >70 μm and <130 μm
- Contamination: No contamination observed during long-term antibiotic and antimycotic free culture

NUMBER OF REPLICATE TISSUES: Three tissues per condition (n = 3)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: A preliminary test was undertaken to check for MTT interference and water colouration (pass confirmed), followed by one main test to obtain the final result

ACCEPTANCE CRITERIA
- Negative control (treated with DPBS): Mean OD570 ≥0.8 and ≤2.8
- Positive control: Mean relative percentage viability after 1 hour ≤20 % of the mean of the negative controls
- Standard deviation of triplicate OD values for triplicate skin models in each experimental condition: <18%
- Blank: Mean OD570 of the 6 wells containing extraction solvent alone (n = 3) ≤0.1

PREDICTION MODEL
- Irritant (I): Test items that reduce viability to 50 % or below
- Non-irritant (NI): Test items with a percentage viability above 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: Single topical application of 30 µl of test item (100% purity) to the surface of EpiDerm™ model
- Concentration: Neat

NEGATIVE CONTROL
- Amount applied: Single topical application of 30 µl of sterile Dulbecco's Phosphate Buffered Saline (DPBS)
- Concentration: Neat

POSITIVE CONTROL
- Amount applied: Single topical application of 30 µl of sodium dodecyl sulphate (SDS)
- Concentration: 5 % in water solvent

Duration of treatment / exposure:

Application of test, positive control, and negative control material for 25 minutes at room temperature (extended by 4 minutes) and 35 minutes at 37 °C, 5 % CO2, ≥95 % RH

Duration of post-treatment incubation (if applicable):

42 ± 4 hours prior to the MTT endpoint

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1)

Value:

34.233

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: Compatability checks (as per SOP L0029) confirmed that the test item did not interfere with MTT.
- Colour interference with MTT: No water colouration was observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Pass, 1.420
- Acceptance criteria met for positive control: Pass, 5.786
- Acceptance criteria met for variability between replicate measurements: Pass, negative control: 3.896; positive control: 0.794; test item: 6.643
- Acceptance of criteria met for blank: Fail, 0.166. Optical density (OD) values obtained with blanks were higher than 0.1 (0.166) causing a deviation from the acceptance criteria. However, the spectrophotometer was fully validated and had passed all required tests. The OD values for blanks observed in this study are consistent with historical data using this spectrophotometer in the XCellR8 laboratory and meet our current internal acceptance criteria of blank OD values <0.194 (mean of XCellR8 historical data, based on blanks obtained during 66 historical runs). Subsequently, the fail result is not considered to be an issue in the interpretation of this study data. This SOP and guideline deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained.

Table 1. Mean and standard deviation (SD) of cell viability measurements and of viability percentages after 65 minutes of application and 42 ± 4 hours post-incubation.

Name	Mean OD	SD OD	Mean viability (%)	SD viability (%)	CV (%)	Classification
Negative control (DPBS)	1.420	0.055	100.000	3.898	3.898	Non-Irritant
Positive control (SDS 5 %)	0.082	0.011	5.786	0.794	13.715	Irritant
Test item (Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1)	0.486	0.094	34.233	6.643	19.405	Irritant

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Relative mean tissue viability (as a percentage of the negative control) obtained for the reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) was 34.233 % after a 65 minute exposure period. The registration substance is subsequently regarded as a Category 2 irritant in accordance with CLP Regulation (EC) No. 1272/2008.

Executive summary:

In line with OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method) and Good Laboratory Practise (GLP), an in vitro skin irritation test was undertaken for the reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1). The test is designed to assess the irritation potential of a test item using the EpiDerm™ Human Skin Model after an exposure period. Of the test item, positive control, and negative control, a single topical application of 30 µl was administered to the epidermal tissue surface in triplicate. The positive control was sodium dodecyl sulphate at a concentration of 5 % in water and the negative control was sterile Dulbecco's Phosphate Buffered Saline at a neat concentration. At the end of the 60 ± 1 minute (extended by 4 minutes) exposure period, epidermal tissues were rinsed and incubated for 42 ± 4 hours. An MTT viability assay was performed with the aid of a spectrophotometer (BMG LabTech FluoStar Optima) at a wavelength of 570 nm for optical density (OD) measurement with no reference filter. All acceptance criteria were met except for the blank mean OD that was >0.1 at 0.166. However, it was determined by the Study Director that this minor deviation in SOP and OECD Guideline protocol would not affect the integrity or interpretation of the results as no equivocal results were observed. In a preliminary test, it was confirmed that the reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) did not interfere with or reduce MTT, nor induce water colouration.

Data are presented in the form of percentage of viability. The positive and negative control had a relative mean tissue viability (calculated as a percentage of the negative control) of 5.768 and 100.000 %, respectively. The viability of the reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) was determined to be 34.233 %. As the percentage viability of the epidermal tissue post-exposure is <50 %, the registration substance can be considered to be irritating to the skin (Category 2) in line with CLP Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 October 2017 to 26 October 2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Corneas from bovine eyes were obtained from a local abattoir. The eyes were removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) in a suitably sized container and transported on the same day to the testing facility.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

Ten minutes

Duration of post- treatment incubation (in vitro):

Two hours

Number of animals or in vitro replicates:

Three corneas (triplicate)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Upon arrival at the test facility, the corneas were excised from the eyes and loaded onto the specifically designed holders . Both chambers of each holder were filled with pre-warmed Minimal Essential Medium (MEM), with the posterior chamber filled first, ensuring that no bubbles were formed. The holders were incubated at 32±1°C for at least 1 hour. After the incubation, the media was removed from both the anterior and posterior chambers. Fresh media was added to the posterior chamber first and then the anterior chamber (this media replacement order ensured the cornea retained its natural curvature as much as possible). The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

QUALITY CHECK OF THE ISOLATED CORNEAS
On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

NUMBER OF REPLICATES
Three.

NEGATIVE CONTROL USED
0.9% sodium chloride solution

POSITIVE CONTROL USED
Dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME
A volume of 750 µL of the test article (or enough test article to completely cover the cornea) was applied to each of three corneas followed by a ten minute incubation at 32±1°C

POST-INCUBATION PERIOD: Two hours

REMOVAL OF TEST SUBSTANCE
After the incubation, each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometer (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
The test article is concluded as inducing serious eye damage if the IVIS is >55.
The test article is concluded as not requiring classification for eye irritation if the IVIS is ≤3.
No prediction can be made if the IVIS is >3 but ≤55.

Irritation parameter:

in vitro irritation score

Value:

20.69

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test article were noted to be cloudy and slightly wrinkled following treatment

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Negative control yields opacity and permeability values that are less than the established upper limits for these endpoints for bovine corneas as treated at the testing facility
- Acceptance criteria met for positive control: Positive control yields an IVIS within 2 standard deviations of the historical control mean.

See attached brackground documents.

Interpretation of results:

other: Inconclusive

Conclusions:

The test article, Reaction mass of morpholine and 6 [(p tosyl)amino]hexanoic acid, compound with morpholine (1:1) (EC 944 090 8), produced an IVIS score of 20.69 and therefore no prediction can be made in respect of it’s potential to cause eye irritation as the IVIS was >3 but <55.

Executive summary:

An in vitro Bovine Corneal Opacity and Permeability (BCOP) assay was performed in line with OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method).

 

The undiluted test material was applied to three cattle corneas obtained from an abattoir at a volume of 750 μL, after which each cornea was incubated at 32 ± 1 °C for 10 minutes and then washed with phenol red-containing media followed by a media without phenol red. Corneal opacity was measured after a 2 hour period of horizontal incubation. For permeability, the corneas were incubated in the vertical position for 1 hour and 25 minutes at 32 ± 1°C within a sodium fluorescein solution. Thereafter, three 350 μL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490). A positive (dimethylformamide) and negative (0.9% sodium chloride solution) control was applied using the same procedure to additional groups of corneas.

 

The mean corrected opacity reading for the test article, positive control, and negative control was 16.0, 62.7, and 0.0, respectively. The mean group corrected optical density for the test article was 0.180 and the mean group corrected optical density for the positive and negative control was 0.243 and 0.000, respectively. An In Vitro Irritation Score (IVIS) of 20.69 was calculated for Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) from corneal opacity and permeability measurements. No prediction can be made if the IVIS is >3 but ≤ 55 and, therefore, the study is inconclusive.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

GLP-compliant studies were performed in order to investigate the skin corrosion and skin irritation of Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) in accordance with the OECD Testing Guidelines 431 and 439 respectively. The substance met the criteria for classification as Skin Irrit 2 according to Regulation (EC) N° 1272/2008.

 

A GLP-compliant study was performed in order to investigate the eye irritation of Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) in accordance with the OECD Testing Guideline 437 (BCOP). The study results do not provide enough information to make a conclusion on the potential for the substance to induce eye irritation.

According to ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 6.0 - July 2017), the BCOP possesses a good predictivity to identify substances inducing serious eye damage and therefore meeting the criteria for classification as Eye Dam. 1 according to Regulation (EC) N° 1272/2008. Since Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) was not identified during the study as inducing serious eye damage, it can be concluded that the substance does not meet the criteria for classification as Eye Dam. 1 according to Regulation (EC) N° 1272/2008.

Therefore it is proposed to classify Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) as Eye Irrit 2 without performing additional in vitro eye irritation testing as it represents a worst-case scenario.