1,1'-[iminobis(ethyleneiminoethylene)]bis[3-(octadecenyl)pyrrolidine-2,5-dione]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 December 2015 to 10 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

1,1'-[iminobis(ethyleneiminoethylene)]bis[3-(octadecenyl)pyrrolidine-2,5-dione]

EC Number:

264-637-8

EC Name:

1,1'-[iminobis(ethyleneiminoethylene)]bis[3-(octadecenyl)pyrrolidine-2,5-dione]

Cas Number:

64051-50-9

Molecular formula:

C52H95N5O4

IUPAC Name:

3-octadecyl-1-[2-({2-[(2-{[2-(3-octadecyl-2,5-dioxopyrrolidin-1-yl)ethyl]amino}ethyl)amino]ethyl}amino)ethyl]pyrrolidine-2,5-dione

Test material form:

liquid: viscous

Details on test material:

- Description: Brown viscous liquid
- Storage Conditions: Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other:

Cell source:

other: EPIDermTM reconstructed human epidermis model

Details on animal used as source of test system:

SOURCE ANIMAL
Model: Reconstructed human epidermis model kit
- Supplier: MatTek
- Batch number: 23306

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 23306

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
PRE-INCUBATION
The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labelled 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 use as a “holding plate” 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) was dispensed into each well. The two plates were placed into the incubator until required.

After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of 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.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS 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 was complete, the inserts were blotted and transferred to labeled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates were placed into a refrigerator overnight, to allow extraction to proceed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 50 µL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.

- Incubation time: 1 hour
- Spectrophotometer: 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 labelled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency was measured using the Anthos 2001 microplate reader.
- Wavelength: 562 nm

TEST FOR DIRECT MT REDUCTION
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:

50 µL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.

NUMBER OF REPLICATE TISSUES: Duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: Killed
- Procedure used to prepare the killed tissues: Freeze-killed tissues were prepared 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 maintenance medium for approximately 1 hour at room temperature.

In addition to the normal test procedure, the MTT reducing test item was applied to two freeze killed tissues. In addition, two freeze killed tissues remained untreated. The untreated freeze killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

- N. of replicates: Two
- Method of calculation used: The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60 Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (mean OD562 of test item / mean OD562 of negative control) x 100

PREDICTION MODEL / DECISION CRITERIA
- 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 applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight):50 µL

Duration of treatment / exposure:

3 minute and 60 minute exposure periods.

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Duplicate tissues were treated with the test material, negative and positive control.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure period

Value:

102.8

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure period

Value:

111.1

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Direct-MTT reduction: An assessment found the test material was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed during the determination of skin irritation potential. However, the results obtained showed a negligible degree of interference (6.1 % relative to the negative control after 3 minutes exposure and 5.0 % relative to the negative control after 60 minutes exposure) due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results.

3 minutes exposure:
Mean of test material treated killed tissues (tkt) = 0.246 OD562
Mean of untreated killed tissues (ukt) = 0.132 OD562
The direct reduction by the test material relative to the negative control value: (0.246 (tkt) – 0.132 (ukt)) / 1.884 (mean of negative control) = 6.1 %

60 minutes exposure:
Mean of test material treated killed tissues (tkt) = 0.245 OD562
Mean of untreated killed tissues (ukt) = 0.150 OD562
The direct reduction by the test material relative to the negative control value:
(0.245 (tkt) – 0.150 (ukt)) / 1.886 (mean of negative control) = 5.0 %


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 1.884 for the 3 Minute exposure period and 1.886 for the 60 Minute exposure period.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.7 % relative to the negative control following the 3 Minute exposure period and 5.1 for the 60 minute exposure period.
- Acceptance criteria met for variability between replicate measurements: In the range 20-100 % viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30 %. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1: Mean OD562Values and Viabilities for the Negative Control Item, Positive Control Item and Test Material

Tissue	Exposure Period	Mean OD562 of individual tissues	Mean OD562 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	2.066	1.884	0.257	13.7	100*
		1.702
	60 Minutes	1.786	1.886	0.141	7.5
		1.985
Positive Control	3 Minutes	0.109	0.107	0.003	N/A	5.7
		0.105
	60 Minutes	0.091	0.096	0.006	N/A	5.1
		0.100
Test Item	3 Minutes	1.893	1.938	0.063	3.2	102.8
		1.982
	60 Minutes	2.082	2.095	0.018	0.8	111.1
		2.107

OD = Optical density

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

Assessment of Colour Interference with MTT

The solution containing the test material did not become coloured. This was taken to indicate the test material did not have the potential to cause colour interference.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test material was determined to be non-corrosive to the skin.

Executive summary:

A study was performed in vitro to assess the corrosive potential of the test material in accordance with the standardised guidelines OECD 431 and EU Method B.40bis under GLP conditions.

Duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. The test employed the use of the EpiDerm™ Human Skin Model. Negative and positive control groups were treated in duplicate for each exposure period. The test material was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test material was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

The mean OD562 for the negative control treated tissues was 1.884 for the 3 Minute exposure period and 1.886 for the 60 Minute exposure period. The relative mean tissue viability for the positive control treated tissues was 5.7 % relative to the negative control following the 3 Minute exposure period and 5.1 for the 60 minute exposure period. The Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied. The relative mean viability of the test material treated tissues was 102.8 for the 3 minute exposure period and 111.1 for the 60 minute exposure period. The relative mean tissue viability (% of negative control) was greater than 50 % at 3 minutes and greater than 15 % at 60 mins, therefore under the conditions of this study, the test material was determined to be non-corrosive to the skin.