Reaction products of fatty acids, C18 (unsaturated) alkyl and epoxidized fatty esters, with amines polyethylenepoly-, tetraethylenepentamine

Endpoint summary

Description of key information

The test item was considered to be corrosive to skin and bassis of this result it is also considered as an eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 23 March 2021 and Experimental completion date: 27 March 2021

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:

adopted: 18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell source:

other: EpiDerm™ Reconstructed Human Epidermis Model Kit

Details on animal used as source of test system:

Not applicable

Justification for test system used:

This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium.
Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit.
Supplier: MatTek In Vitro Life Sciences Laboratories
Date received: 24 March 2021
EpiDermTM Tissues (0.63cm2) lot number: 34137
Assay Medium lot number : 031821LHC
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.

Main test.
The assay medium was brought to room temperature before use.
0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 Minute and 60 Minute exposure periods.
EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium.
The 6 well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing
Before pre-incubation was complete, a 24 well plate was prepared for both the 3 Minute and 60 Minute exposure periods.
This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT-loading.
Another 24 well plate was prepared for the MTT-loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT-loading plate.

After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced fresh assay medium.
The 6-well plate for the 3 Minute exposure period was returned to the incubator, while the other was being dosed for the 60 Minute exposure. For the 60 Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period.

When dosing for the 60 Minute exposure period was complete, the same procedure was repeated for the 3 Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24 well plate prepared for MTT-loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated.

After the 3 Hour MTT incubation, the tissue were blotted and transferred to 24 well plates for formazan (reduced MTT) extraction.
The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol.
The plate was covered with plate sealer, to prevent isopropanol evaporation, and stood overnight at room temperature, to allow extraction to proceed.

Absorbance/Optical Density Measurements
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader and LT-com analysis software.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test item: 50 µL
Negative control: 50 µL
Positive control: 50 µL (8.0 N)

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

24 well plate was prepared for use as a “holding plate” for both the 3 Minute and 60 Minute exposure periods.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Exposure period: 3 minutes

Value:

41.8

Vehicle controls validity:

not applicable

Remarks:

test item used as supplied

Negative controls validity:

valid

Remarks:

100% viability

Positive controls validity:

valid

Remarks:

3.0% viability

Remarks on result:

other: corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Exposure period: 60 minutes

Value:

34.4

Vehicle controls validity:

not applicable

Remarks:

test item used as supplied

Negative controls validity:

valid

Remarks:

100% viability

Positive controls validity:

valid

Remarks:

3.2% viability

Remarks on result:

other: corrosive

Other effects / acceptance of results:

ACCEPTANCE CRITERIA

Negative control
The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.

Positive control
Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60‑Minute positive control is < 15%.

Coefficient of Variation
In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

RESULTS:
The mean OD570 for the negative control treated tissues was 2.011 for the 3 Minute exposure period and 1.997 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 3.2% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
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:

Category 1 (corrosive) based on GHS criteria

Conclusions:

The test item was considered to be corrosive: UN GHS H314 Combination of sub‑categories 1B and 1C.

Executive summary:

Introduction

The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. Viable cells are able toreduce MTT to formazanwhereas non-viable cells cannot. The results are used to make a prediction of the corrosivity potential of the test item (increased cytotoxicity is indicative of corrosion potential).

Methods

Duplicate tissues were treated with the negative control, positive control and test item for exposure periods of 3 and 60 minutes. As the test item had been shown to directly reduce MTT additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. At the end of the exposure period the control items and test item were rinsed from the tissues before each tissue was taken for MTT‑loading. After MTT-loading the tissues were placed into 2 mL of isopropanol for formazan extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD₅₇₀).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	3.0	41.8
60 minute	100*	3.2	34.4

*The mean viability of the negative control tissues is set at 100%

Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

Conclusion

In this study and under the experimental conditions reported:

The test item was considered to be corrosive: UN GHS H314 Combination of sub‑categories 1B and 1C.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (Category 1)

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The substance is classified as corrosive to the skin, so no eye irritation study is necessary and the substance will be classified appropriately.

Justification for classification or non-classification

Based on the findings in the skin corrosion study the test substance considered to be classified as corosive to skin according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and ultimately considered to be irritating to the eyes.