[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 February to 05 March 2021

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:

June 26, 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EpiDermTM (EPI-200-MatTek Corporation)

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Initially, the predictive capacity of the modified EpiDerm™ Skin Irritation Test (SIT) test method using MatTek EpiDermTM 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 methods 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, 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:

The reconstructed human epidermal model EpiDermTM (EPI-200-MatTek Corporation) was used.

MatTek’s EpiDermTM model has been extensively characterised for multiple parameters including morphology, tissue viability, skin barrier function and sterility (for information see www.mattek.com). QC results for the specific lot of models received (Lot# 34130) were checked in-house for compliance with MatTek acceptance ranges with the following outcome (details in Annex 1):

• Morphology - PASS
• Tissue viability - PASS
• 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)- PASS
• Sterility testing showed no contamination during long term antibiotic and antimycotic free culture- PASS

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µl of neat test item, negative control (DPBS) or positive control (SDS 5%)

Duration of treatment / exposure:

60±1 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, in humidified conditions)

Duration of post-treatment incubation (if applicable):

42±4 hours post-treatment incubation, prior to the MTT endpoint; three tissues per condition (n=3).

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

relative to negative control

Run / experiment:

Run 1 (3 replicates) 60±1 minute exposure on the surface of the EpiDermTM reconstructed human epidermis, and a 42 ±4 hour post-exposure incubation time.

Value:

46.604

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

All acceptance criteria indicated in the method section were passed

Table: Mean and SD of cell viability measurements and of viability percentages after 60±1 minutes of application and 42±4 hours post-incubation

 

Name	Code	Mean of OD	SD of OD	Mean of viability (%)	SD of viability (%)	CV %	Classification
DPBS	NC	1.674	0.032	100.000	1.900	1.900	Non-Irritant
SDS 5%	PC	0.081	0.024	4.860	1.442	29.670	Irritant
N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride	TA6	0.780	0.182	46.604	10.880	23.346	Irritant

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

A test item is considered an irritant (I) to skin in accordance with UN GHS Category 2 if the skin model viability after exposure and post-treatment incubation is ≤50%.

Conclusions:

The percentage of viability obtained with the test item N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride was 46.604%, therefore it was considered as Irritant to the skin.

Executive summary:

In this study, the skin irritation potential of N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride was assessed in vitro according to OECD Test Guideline 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.

After 60±1 minute exposure on the surface of the EpiDerm^TM reconstructed human epidermis, and a 42 ±4 hour post-exposure incubation time, viability of the tissues was assessed and compared to the negative control.

The percentage of viability obtained was 46.604% and therefore: N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride was classified as Irritant to the skin.

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:

11 May 2021 (1 day)

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)

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Due to the short-term nature of the study no analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the integrity or validity of the study.

Specific details on test material used for the study:

For the purpose of this study the test item was ground using a mortar and pestle and prepared as a 20% w/v solution in sodium chloride 0.9% w/v.

The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

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.

Species:

cattle

Details on test animals or tissues and environmental conditions:

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.

Vehicle:

other: The test item was ground using a mortar and pestle and prepared as a 20% w/v solution in sodium chloride 0.9% w/v.

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

The test item was ground using a mortar and pestle and prepared as a 20% w/v solution in sodium chloride 0.9% w/v.

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

A post-treatment opacity reading. The permeability of the corneas to sodium fluorescein after 90 minutes.

Number of animals or in vitro replicates:

triplicate

Details on study design:

NUMBER OF REPLICATES
triplicate

NEGATIVE CONTROL USED
Sodium chloride 0.9% w/v

POSITIVE CONTROL USED
Imidazole

APPLICATION DOSE AND EXPOSURE TIME
20% w/v; 240 minutes

TREATMENT METHOD:
Bovine Corneal Opacity and Permeability (BCOP) holders.

POST-INCUBATION PERIOD:
A post-treatment opacity reading. The permeability of the corneas to sodium fluorescein after 90 minutes.


REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
the cornea was rinsed 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.

- POST-EXPOSURE INCUBATION:
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
A post-treatment opacity reading was taken and each cornea was visually observed.

- Corneal permeability:
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated.


- Others
Histopathology


SCORING SYSTEM:
In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
For an acceptable test the following positive control criterion should be achieved:
20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during the previous 12 months for this testing facility.
For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values, or an overall IVIS value of < 3 calculated from the previous 12 months data for this testing facility.

Irritation parameter:

in vitro irritation score

Value:

122.5

Negative controls validity:

valid

Remarks:

0.7

Positive controls validity:

valid

Remarks:

78.3

The positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.
The negative control values were within accepted limits. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

According to UN GHS Classification, the test item N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride causes serious eye damage and is classified as Category 1 under the conditions of the test.

Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS). Test items inducing serious eye damage are classified as UN GHS Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS No Category.

 

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

 

The In Vitro irritancy score for the test item was 122.5. Positive and negative control results were valid. According to UN GHS Classification, the test item N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride causes serious eye damage and is classified as Category 1 under the conditions of the test.

 

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Justification for classification or non-classification

The skin irritation potential of the test substance was assessed in vitro according to OECD Test Guideline 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. The percentage of viability obtained was 46.604%. Under CLP, the substance is skin irritant (category 2).

 

Eye irritation test conducted according to OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method was inconclusive. In the BCOP test conducted according to OECD 437, the in Vitro irritancy score for the test item was 122.5. Positive and negative control results were valid. Under the CLP, the test substance is classfied as category 1 (serious eye damage).