N,N'-bis(1,3-dimethylbutylidene)ethylenediamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-11-10 to 2021-11-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-06-18

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

amended 2019-07-31

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(1,3-dimethylbutylidene)ethylenediamine

EC Number:

247-196-6

EC Name:

N,N'-bis(1,3-dimethylbutylidene)ethylenediamine

Cas Number:

25707-70-4

Molecular formula:

C14H28N2

IUPAC Name:

N,N'-bis(1,3-dimethylbutylidene)ethylenediamine

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

other: human adult donor

Justification for test system used:

The corrosivity potential of a chemical may be predicted by measurement of its cytotoxic effect, as reflected in the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] assay, on the EpiDerm™ reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of corrosives in the in vivo Rabbit skin assay (OECD 404) and is specifically approved as a replacement for the in vivo skin corrosivity test within OECD 431.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, MA, USA and Bratislava, Slovakia)
- Tissue batch number: 36103
- Shipping date: 08 November 2021
- Delivery date: 09 November 2021
- Date of initiation of testing: 12 November 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure: 37±1 °C
- Temperature of post-treatment incubation: 37±1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing steps:
After the exposure time the EpiDerm™ units were removed and rinsed thoroughly with 1x DPBS sterile solution to remove all of the test material from the epidermal surface. The constant soft stream of DPBS was used from approximately 1.5 cm distance, and filling and emptying the tissue insert was repeated 20 times. The rest of the DPBS was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle and touching the upper lip to the absorbent material (to break the surface tension).
Following rinsing, the tissues were transferred and kept in the 24-well holding plates (pre-filled with 300 μL medium) until the rinsing procedure was completed. The rest of the DPBS was removed from the tissues by gently sweeping the tissues’ surface with a sterile cotton tipped swab (care was taken to avoid the damage of tissues).
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL MTT per well
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Varioskan™ LUX Type 3020
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 200 – 1000 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
The EpiDerm™ System is manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium are tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek determines the ET-50 value following exposure to Triton X-100 (1%) for each EpiDerm™ lot. The ET-50 must fall within a range established based on a historical database of results.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues: for the MTT evaluation, batch No. of killed epidermis: 34139
- N. of replicates: 3
- Method of calculation used: See "Any other information on materials and methods, incl. tables"

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability is < 50 % after 3 min exposure OR ≥ 50 % after 3 min exposure AND < 15 % after 60 min exposure.
- The test substance is considered to be non-corrosive to skin if the viability is ≥ 50 % after 3 min exposure AND ≥ 15 % after 60 min exposure

If the test substance is considered to be corrosive, then the following criteria apply:
- If the cell viability is < 25 % after 3 min exposure, then the the test substance is clasified as optional Sub-category 1A
- If the cell viability is ≥ 25 % after 3 min exposure, then the the test substance is clasified as a combination of optional Sub-categories 1B-and-1C

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

60 and 3 minutes

Duration of post-treatment incubation (if applicable):

MTT assay: 3 hours (± 5 min);
The exposure of test item was terminated by rinsing with 1xDPBS, the EpiDerm™ units were transferred into the MTT ready to use solution filled 24-well plate (300 μL of 1 mg/mL MTT per well) and then incubated at 37±1 °C in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere.

Number of replicates:

for both exposure times: 3 replicates of the test item, 3 replicates of negative control and 3 replicates of positive control were used. Furthermore, 3 killed test item-treated tissues and 3 killed negative control-treated tissues were used for the MTT evaluation for each exposure time.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 / 3 min

Value:

64

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1/ 60 min

Value:

10

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The mean OD value of the three negative control tissues was 2.038 and 2.140 at 60 and 3 minutes exposure respectively. The mean OD value obtained for the positive control was 0.104 at
60 minutes exposure which result corresponds to 5 % viability, when compared to the results obtained from the negative control. Each calculated Coefficient of Variation value (CV) in the range of 20-100 % viability was below 30. The mean OD value of the blank samples MTT-100-EXT were 0.0375 and 0.0376 (below 0.1).
All validity criteria were within acceptable limits and therefore the study was considered to be valid.

The test item interacted with the MTT, therefore additional controls and data calculations were necessary.
The mean non-specific MTT reduction (NSMTT) was determined to be 2 % and 0 % at the 60 min and 3 min exposure respectively. As the NSMTT were below 30 % in each case. However, the true MTT metabolic conversion was made and the correction of viability percentages were undertaken after the 60 minutes exposure, because the correction with 0% was not interpretable at the 3 minutes exposure.

Any other information on results incl. tables

OD values and viability percentages of the controls:

Controls	Optical Density (OD)				Viability (%)
Negative Control: Sterile deionized water 60 min exposure	1	2.139			105
	2	1.996			98
	3	1.980			97
	mean	2.038			100
	SD				4.303
	CV				4.303
Negative Control: Sterile deionized water 3 min exposure	1	2.100			98
	2	2.319			108
	3	2.000			93
	mean	2.140			100
	SD				7.630
	CV				7.630
Positive Control: Potassium hydroxide (KOH) 8N solution 60 min exposure	1	0.129			6
	2	0.068			3
	3	0.114			6
	mean	0.104			5
	SD			1.560
	CV			30.607
Positive Control: Potassium hydroxide (KOH) 8N solution 3 min exposure	1	0.282			13
	2	0.546			26
	3	0.325			15
	mean	0.384			18
	SD			6.627
	CV			36.902

 

OD values and viability percentages of the test item (including corrected values):

Substance	Optical Density (OD)		TODTT	Viability (%)	Relative Viability (%)
Test Item: 60 min exposure	1	0.216	0.185	11	9
	2	0.191	0.160	9	8
	3	0.322	0.291	16	14
	mean	0.243	0.212	12	10
	SD			3.413	3.413
	CV			28.654	32.843

 

OD values and viability percentages of the test item:

Test Item	Optical Density (OD)		Viability (%)
Test Item: 3 min exposure	1	1.486	69
	2	1.239	58
	3	1.383	65
	mean	1.369	64
	SD		5.805
	CV		9.072

 

 

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

In conclusion, in this in vitro skin corrosion test in EpiDermTM model (OECD 431) with the test sbstance, the results indicate that the test item is corrosive. According to the UN GHS classification systems, the test sbstance has been categorized as “Corrosive: A combination of optional sub-categories 1B and 1C”.

Executive summary:

EpiDerm^TM test of the test item has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431, 18 June 2019. Disks of EpiDerm^TM (three units / chemical / incubation time) were treated with the test item and incubated for 60 minutes (+ 3 min) at standard culture condition (37±1 °C in an incubator with 5±1 % CO₂ in a 95 % humidified atmosphere) and 3 min at room temperature. Exposure of test material was terminated by rinsing with 1x DPBS solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±5 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO₂ in a 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using MTT-100-EXT and quantified spectrophotometrically. Potassium hydroxide (KOH) 8N solution and sterile deionized water treated (three units / positive and negative control) epidermis were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to the negative control.

The test item is a MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement.

For each treated tissue, viability was expressed as % relative to the negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 60 minutes of exposure is above or equal 15 % and the mean relative viability after 3 minutes of exposure is above or equal 50 % of the negative control.

The test item showed significantly reduced cell viability in comparison to the negative control after 60 minutes of exposure. However, the test item did not show significantly reduced cell viability in comparison to the negative control after 3 minutes of exposure. The average test item treated tissue relative viability was 10 % at 60 minutes of exposure and the mean viability was 64 % at 3 minutes of exposure.

Positive and negative controls showed the expected optical density (OD) and cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid.

In conclusion, in this in vitro skin corrosion test in EpiDerm^TM model (OECD 431) with ANCAMINE 2458, the results indicate that the test item is corrosive. According to the UN GHS classification systems, the test sbstance has been categorized as “Corrosive: Optional Sub- categories 1B and 1C”.