1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts

Endpoint summary

Description of key information

Skin Corrosion

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy- N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts was tested according to OECD Test Guideline 431 to predict potential for skin corrosivity by cytotoxicity in the EpiDerm™ tissue. Following a 60 minute exposure period to the test item, mean tissue viability was 80.8%, well above the <15% threshold for classification as corrosive. Therefore, it was concluded the substance is not corrosive to the skin according to CLP Regulation (EC) 1272/2008.

 

Skin Irritation

Following the results of the skin corrosion test, 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy- N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts was tested according to OECD Test Guideline 439 to predict potential for skin irritation using the EPISKIN™ tissue model. Following a 15 minute exposure period to the test item, mean tissue viability was 87.0%, above the >50% threshold for classification as irritant. Therefore, it was concluded the substance is not an irritant to the skin according to CLP Regulation (EC) 1272/2008.

 

Eye Damage

A GLP-compliant in vitro eye damage study was performed on the substance in accordance with the OECD Testing Guideline 437. This test method can correctly identify test items inducing serious eye damage (IVIS > 55) as well as those not requiring classification for eye irritation or serious eye damage (IVIS ≤ 3). However, due to high false positive rates, it should not be the first-choice method for a bottom-up approach. No stand-alone prediction can be made for IVIS scores >3 and ≤ 55.

The IVIS score obtained in this test was 15.1 (mean corneal opacity value was 10.0) and, therefore, no prediction could be made, and a subsequent test was commissioned in accordance with the OECD Testing Guideline 429.

 

Eye Irritation

A GLP compliant study was performed to assess the eye irritation potential of the substance using the human cornea model EpiOcular™, in accordance with OECD Test Guideline 492. The EpiOcular™ can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. However, it does not allow discrimination between serious eye damage/irreversible effects on the eye (Category 1) and eye irritation/reversible effects on the eye (Category 2), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B), as defined by UN GHS.

If the test item-treated tissue viability is > 60% relative to the negative control-treated tissue viability, the test item is labelled as UN GHS No Category. If mean tissue viability is < 60%, no prediction can be made from this result in isolation because in case of a true positive, the methods cannot resolve between UN GHS Categories 1 and 2.

The result, mean tissue viability, was 3.194 %. According to the result, the test item cannot be labelled as UN GHS No Category.

Based on results of OECD 437, no stand-alone prediction can be made. However, results of OECD 492 provide supporting evidence that the test item cannot be labelled as UN GHS No Category. As OECD 492 in isolation does not allow discrimination between UN GHS Category 1 and UN GHS Category 2, Defined Approaches 1 (DAL-1) from OECD TG 467 (2022) were used to discriminate between UN GHS Category 1 and UN GHS Category 2. Based on water solubility of 520 mg/ml, OECD TG 492 VRM 1 cell viability < 60% and OECD TG 437 opacity < 145, the test item is classified as UN GHS Category 2.

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:

02 May, 2018 - 04 May, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

yes

Remarks:

An assessment found the test item was able to directly reduce MTT and an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DSP-001.
- Expiration date of the lot/batch: 15 August 2018.
- Purity test date: No data.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item (solid) was pressed flat and appropriate sized discs were cut out. The application of the discs is an acceptable method in accordance with the MatTek SOP MK-24-007-0024 of the validated and regulatory accepted EpiDerm SCT.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit.
- Supplier: MatTek.
- Tissue batch number(s): 28602.
- Delivery date: 01 May 2018.
- Date of initiation of testing: 02 May 2018.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C.
- Temperature of post-treatment incubation (if applicable): Room temperature.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL.
- Incubation time: 3 hours.
- Spectrophotometer: Labtech LT-4500 microplate reader.
- Wavelength: 570 nm.
- Linear OD range of spectrophotometer: 0-4 OD.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: ET-50 fall within the range of the 1996 EpiDerm database of 4.77 – 8.72 hours.
- Barrier function: Lipid precursors used to enhance epidermal barrier formation.
- Morphology: Good retention of epidermal morphology for up to 3 weeks.
- Contamination: All tissues are visually inspected and if physical imperfections are noted, tissues are rejected for shipment.
- Reproducibility: Tissues may be stored at 4°C for up to 6 days prior to use, although best reproducibility obtained if used on same day.

NUMBER OF REPLICATE TISSUES: 2.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : The test item was shown to directly reduce MTT in the direct MTT reduction test, therefore the determination of skin corrosion potential was performed in parallel on viable and freeze-killed tissues.
- Procedure used to prepare the killed tissues: Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERM tissues in an empty 12-well plate and storing in a freezer (−14 to −30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
- Number of replicates: 2.
- Method of calculation used: Relative mean viability (%) - (mean OD570 of test item / mean OD570 of negative control) x 100.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.0931 g.

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL.
- Concentration (if solution): 100%.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL.
- Concentration (if solution): 100%.

Duration of treatment / exposure:

3 and 60 minutes.

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

90

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Percentage viability 100%

Positive controls validity:

valid

Remarks:

Percentage viability 3.3%

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

80.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Percentage viability 100%

Positive controls validity:

valid

Remarks:

Percentage viability 2.9%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None observed.
- Direct-MTT reduction: The results obtained from the freeze-killed tissues showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results for quantitative correction of results or for reporting purposes.
- Colour interference with MTT: The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.854 for the 3-Minute exposure period and 1.927 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 2.9% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy- N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts was tested according to OECD Test Guideline 431 to predict its potential for skin corrosivity by cytotoxicity in the EpiDerm™ tissue. Following a 60 minute exposure period to the test item, mean tissue viability was 80.8%, well above the <15% threshold for classification as corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May, 2018 - 11 June, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: DSP-001.
Expiry Date: 15 January 2019.

Test system:

human skin model

Remarks:

EPISKIN™ Reconstructed Human Epidermis Model Kit.

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit.
- Tissue batch number(s): 18-EKIN-023.
- Delivery date: 5th June 2018.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C.
- Temperature of post-treatment incubation (if applicable): 37 °C.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL.
- Incubation time: Incubated in the dark at 37 °C, 5% CO2 in air for 3 hours.
- Spectrophotometer: Labtech LT-4500 microplate reader.
- Wavelength: 570 nm.

NUMBER OF REPLICATE TISSUES: Triplicate (3).

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
The test was repeated twice due to a failure to meet the assay acceptance criteria and once due to a technical issue.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the viability after 15 minutes exposure less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure is greater than 50%.
If the relative mean tissue viability is ≤50%, differentiation between EU CLP/UN GHS Category 1 and Category 2 will not be possible based on the results of this study.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The test item was pressed flat and appropriate sized discs of a similar diameter to the tissues were cut out with approximate weight of 0.0455g.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL applied to triplicate tissues.
- Concentration (if solution): as supplied.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL applied to triplicate tissues.
- Concentration (if solution): 5% w/v aqueous solution.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

Rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.

Number of replicates:

Triplicate (3)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

85.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

88.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

87.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None recorded.
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The test was repeated twice due to a failure to meet the assay acceptance criteria and once due to a technical issue.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD 570 for the negative control treated tissues was 0.829 and the standard deviation value of the viability was 7.1%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 4.9% relative to the negative control treated tissues and the standard deviation value of the viability was 1.0%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.8%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean viability of the test item treated tissues was 87.0% after a 15-minute exposure period and 42-hour post-exposure incubation period. As this value is greater than 50%, the substance is not classified for irritation.

Executive summary:

A GLP compliant study was performed to assess the skin irritation potential of the substance using the EPISKIN™ Reconstructed Human Epidermis Model, in accordance with OECD Test Guideline 439.

Triplicate tissues were prepared and treated with the test item and positive and negative controls for a 15 minute period before incubation for 42 hours.

The optical density (OD₅₇₀) was measured using a microplate reader and the percentage tissue viabilities recorded.

All acceptance criteria were acheived in test item and controls and there were no deviations from the study plan.

The relative mean viability of the test item treated tissues was determined to be 87.0% and therefore concluding the substance is not classified as irritating to the skin, in accordance with CLP Regulation (EC) 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

14 June, 2018

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)

Remarks:

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Specific details on test material used for the study:

Batch: DSP-001.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Local abbatoir.
- Characteristics of donor animals: 12-60 months old.
- Storage, temperature and transport conditions of ocular tissue: Placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL).
- Time interval prior to initiating testing: Transported to the test facility over ice packs on the same day of slaughter.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL.
- Concentration: The test item was prepared as a 20% w/v solution in sodium chloride 0.9% w/v.

Duration of treatment / exposure:

10 minutes.

Duration of post- treatment incubation (in vitro):

4 hours.

Number of animals or in vitro replicates:

Triplicate (3).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

QUALITY CHECK OF THE ISOLATED CORNEAS

NUMBER OF REPLICATES
3 for test item, 3 for negative control, 3 for positive control.

NEGATIVE CONTROL USED
Sodium chloride 0.9% w/v.

POSITIVE CONTROL USED
Imidazole 20% w/v.

APPLICATION DOSE AND EXPOSURE TIME
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

TREATMENT METHOD: Closed chamber.

POST-INCUBATION PERIOD: Yes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed at least 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

- POST-EXPOSURE INCUBATION: 32 ± 1 ºC for 240 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of Labtech LT-4500 microplate reader (OD490) .

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) .

DECISION CRITERIA:
IVIS = mean opacity value + (15 x mean permeability OD492 value).
Additionally, the opacity and permeability values are evaluated independently to determine whether the test item induced a response through only one of the two endpoints.
The condition of the cornea is visually assessed post treatment and post incubation.
The test item is classified according to the following prediction model:
IVIS Classification:
≤ 3: No category. Not requiring classification to UN GHS or EU CLP;
> 3: ≤55 No prediction of eye irritation can be made;
> 55: Category 1. UN GHS or EU CLP Causes serious eye damage.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

15.1

Negative controls validity:

valid

Remarks:

The negative control gave opacity of ≤2.4 and permeability ≤0.072.

Positive controls validity:

valid

Remarks:

The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3.

Remarks on result:

not determinable

Irritation parameter:

cornea opacity score

Run / experiment:

Test item / 9

Value:

8

Irritation parameter:

cornea opacity score

Run / experiment:

Test item / 10

Value:

15

Irritation parameter:

cornea opacity score

Run / experiment:

Test item / 11

Value:

7

Irritation parameter:

cornea opacity score

Run / experiment:

Negative control / 2

Value:

0.008

Irritation parameter:

cornea opacity score

Run / experiment:

Negative control / 2

Value:

0

Irritation parameter:

cornea opacity score

Run / experiment:

Negative control / 4

Value:

1

Irritation parameter:

cornea opacity score

Run / experiment:

Positive control / 5

Value:

53

Irritation parameter:

cornea opacity score

Run / experiment:

Positive control / 6

Value:

70

Irritation parameter:

cornea opacity score

Run / experiment:

Positive control / 7

Value:

64

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were partly cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied.

Mean (n=3) corrected test item corneal opacity was measured at 10.0.

Mean corrected permeability (OD492) of test item was 0.341.

Interpretation of results:

study cannot be used for classification

Conclusions:

Baed on the results of the study, no prediction of eye effects could be made.

Executive summary:

A GLP-compliant in vitro eye damage study was performed on the substance in accordance with the OECD Testing Guideline 437. The IVIS score obtained was >3 and ≤ 55, therefore, no prediction could be made and a subsequent test was comissioned following OECD Testing Guideline 492.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July, 2018 - 09 August, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 29 June 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch No.: DSP-001.
Expiry date: 15th January 2019.

Species:

human

Strain:

other: Normal human epidermal keratinocytes (NHEK).

Details on test animals or tissues and environmental conditions:

The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.

The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazol 2-yl) 2,5-diphenyl-tetrazolium bromide] into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm diameter).

CERTIFICATE OF ANALYSIS
- Lot number: 27062
- Keratinocyte Strain: 4F1188
- Tissue viability: (OD540-570nm) 2.016±0.011
- Sterility: Sterile (no contamination)
- Date: August 7, 2018

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50±2 mg of the test item applied topically in duplicate.

Duration of post- treatment incubation (in vitro):

6 hours ± 15 minutes.

Number of animals or in vitro replicates:

Duplicates.

Details on study design:

- RhCE tissue construct used:
EpiOcular™ kits purchased from MatTek Corporation (82105 Bratislava, Slovakia).

- Doses of test chemical and control substances used :
Test item: Applied at 50 ± 2 mg to duplicate tissues for 6 hours ± 15 minutes (83.3 mg/cm² according to guideline).
Negative control: Deionised water, 50 µL applied to duplicate tissues for 6 hours ± 15 minutes.
Positive Control: Methyl acetate, 50 µL applied to duplicate tissues for 6 hours ± 15 minutes.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
After the overnight incubation, the tissues will be pre-wetted with 20 µL of Ca2+Mg2+free-DPBS. If the Ca2+Mg2+free-DPBS will not spread across the tissues, the plate may be tapped to assure that the entire tissue surface is wetted. The tissues will be incubated at standard culture conditions for 30 ± 2 minutes.
After the 30 ± 2 minutes Ca2+Mg2+free-DPBS pre-treatment, the test and control item is tested by applying approximately 50 mg (test item) or 50 µL (controls) topically on the EpiOcular™ tissues. The tissues are incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) for 6 hours ± 15 minutes.
At the end of the 6 hours ± 15 minutes treatment time, the test item will be removed by extensively rinsing the tissues with Ca2+Mg2+free-DPBS (brought to room temperature).

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
- Direct MTT Reduction:
Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it is necessary to assess this ability for each test item prior to conducting any assays with viable tissues. For this purpose 50 mg ± 2 mg of the test item are added to 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture is incubated in the dark at 37 ± 1.5°C in a humidified atmosphere of 5 ± 0.5% CO2 in air for approximately three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) should be run concurrently. If the MTT solution colour turns blue/purple, the test item is presumed to have reduced the MTT. Water insoluble test materials may show direct reduction (darkening) only at the interface between the test item and the medium.
In cases where the test item is shown to reduce MTT, a functional check using freeze-killed tissue controls (killed controls = KC) should be performed in at least one definitive assay to evaluate whether the test material is not binding to the tissue and leading to a false MTT reduction signal. Blue, dark purple and black test items should also be tested on freeze-killed tissues because it may not be possible to assess their potential to directly reduce MTT.
- Coloured or Staining Materials:
Coloured test items or test items which become coloured after application to the tissues may interfere with the quantitative photometric MTT measurement if the colorant binds to the tissue and is extracted together with MTT. Therefore, each test item has to be checked for its colorant properties.
For this reason 50 ± 2 mg of the test item are added to 2 mL of isopropanol (glass tube) and shaken for 2 to 3 hours at room temperature, and also 50 ± 2 mg of the test item are added to 1 mL of deionised water (glass tube) and incubated at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for at least 1 hour. If isopropanol or water will not or only slightly be dyed, or if the test item is not dissolved in isopropanol or water, an additional test with viable tissues (but without MTT addition in the MTT assay) does not have to be performed for determination of a correction factor for calculating the true viability in the main experiment.
If isopropanol or water will be dyed intensively (red, yellow, green, blue, purple, black, brown) two additional viable tissues (without MTT-addition) will be exposed to the test item as described above (+ 2 x negative control tissues).
If isopropanol or water will not or only slightly be dyed, or if the test item is not dissolved in isopropanol or water, an additional test with viable tissues (but without MTT addition in the MTT assay) does not have to be performed for determination of a correction factor for calculating the true viability in the main experiment.
If isopropanol or water will be dyed clearly after addition of a slight coloured, a colourless or white test item, the OD of two 200 µL aliquots of the solution and two 200 µL aliquots of neat isopropanol or of water, respectively, will be measured (e. g. Versamax® Molecular Devices, 85737 Ismaning, Germany, Softmax Pro Enterprise, Version 4.7.1) at a wavelength of 570 nm without reference filter. If the test item is not dissolved completely in isopropanol, the mixture should be centrifuged prior to measurement. The supernatant will be used for measurement.
If the difference of the test item OD and the isopropanol OD is > 0.08, a test with additional viable tissues as described above will be performed (+ 2 x negative control tissues).

- Number of tissue replicates used per test chemical and controls:
Duplicates of test item, and negative and positive controls.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) :
The absorbance at 570 nm (OD570) of each well is measured with in a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Softmax Pro Enterprise, Version 4.7.1). No reference wavelength measurement will be used.

- Acceptable variability between tissue replicates for positive and negative controls :
The negative control OD is > 0.8 and < 2.5;
The mean relative viability of the positive control is below 50% of the negative control viability.

- Acceptable variability between tissue replicates for the test chemical:
The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the freeze-killed tissues and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: mean tissue viability

Value:

3.194

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed purple colour, indicating that the test item does reduce MTT. An additional test with freeze-killed tissues was necessary. The test item proved to dye isopropanol in the colour interference pre-test and additional tests with viable tissues had to be performed.

Acceptance criteria:
1) The negative control OD is > 0.8 and < 2.5 (1.858 and 1.983).
2) The mean relative viability of the positive control is below 50% of the negative control viability (33.47%).
3) The difference of viability between the two relating tissues of a single item is < 20% (values between 0.01% and 5.27%) in the same run (for positive and negative control tissues and tissues of single test items). This applied also to the killed controls (items and negative control) and the additional viable tissues (without MTT addition) which were calculated as percent values related to the viability of the relating negative control.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

After treatment with the test item the residual viability of the tissues was 3.194%. This result is ≤ 60% relative to the negative control and therefore the test item has to be considered as irritating for the eye.

Executive summary:

A GLP compliant study was performed to assess the eye irritation potential of the substance using the EpiOcular™ Human Cornea Model Test, in accordance with OECD Test Guideline 492.

Duplicate tissues were treated with the neat test item and positive and negative controls for 6 hours before rinsing, incubabtion (post-soak) in the assay medium and final post-treatment incubation.

OECD 437 and OECD 492 were commissioned in June 2018 and July 2018, respectively, as part of a top‑down approach to identify ocular corrosives and severe irritants (UN GHS Category 1).

Validated methods are used in a two-step procedure to determine if a test material is a severe irritant (UN GHS Cat.1) or non-classified (UN GHS No Cat.). A default UN GHS Cat. 2 classification could be assigned if neither a severe irritancy nor non-classified assignments were made. (Scott et al, 2010).

The optical density (OD₅₇₀) was measured using a microplate reader and the percentage tissue viabilities recorded.

All acceptance criteria were acheived in the test item and controls and there were no deviations from the study plan.

The mean viability for the test item treated cells was determined to be 3.194%. Therefore, 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts has eye irritating potential and should be classified an eye irritant in accordance with CLP Regulation (EC) 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)