Propanenitrile, 3-[[4-[[5,6(or 6,7)-dichloro-2-benzothiazolyl]azo]phenyl]ethylamino]-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-09-23 - 2021-10-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

No. 640/2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UN GHS (2003, last rev. 2015)

Deviations:

no

Principles of method if other than guideline:

MatTek Corporation Protocol: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT); Version 12/04/2020.
MatTek Corporation Protocol: EpiOcularTM Eye Irritation Test (OCL-200-EIT)
For the prediction of acute ocular irritation of chemicals: Identification of chemicals not requiring classification and labeling for eye irritation or serious eye damage; Version 02/02/2021
Note:
The Pre-Test (Assessment of Colored or Staining Materials and Assessment of Direct Test Article Reduction by MTT) was performed according to the MatTek Protocol “EpiOcularTM Eye Irritation Test.
Reason:
The OECD TG 439 requires the evaluation of colored or staining materials spectrophotometrically but does not state the evaluation criteria. These criteria are missing in the MatTek protocol “In vitro EpiDermTM Skin Irritation Test”, too. But the identical scientific hypothesis is to clarify in the Eye Irritation Test and there the requirements are described.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 23 November 2018; 21 February 2019; 12-14 March 2019

Test material

Test material information

1

Reference substance name:

Reaction mass of 3-({4-[2-(5,6-dichloro-1,3-benzothiazol-2-yl)diazen-1-yl]phenyl}(ethyl)amino)propanenitrile and 3-({4-[2-(6,7-dichloro-1,3-benzothiazol-2-yl)diazen-1-yl]phenyl}(ethyl)amino)propanenitrile

EC Number:

616-632-0

Cas Number:

78564-87-1

Molecular formula:

C18H15Cl2N5S

IUPAC Name:

Reaction mass of 3-({4-[2-(5,6-dichloro-1,3-benzothiazol-2-yl)diazen-1-yl]phenyl}(ethyl)amino)propanenitrile and 3-({4-[2-(6,7-dichloro-1,3-benzothiazol-2-yl)diazen-1-yl]phenyl}(ethyl)amino)propanenitrile

Test material form:

solid: particulate/powder

Details on test material:

Identification: Disperse Red 153

In vitro test system

Test system:

human skin model

Source species:

human

Cell source:

other: Standard Assay Kit and MTT-100 kit were purchased from MatTek Corporation (82105 Bratislava, Slovakia, Lot No.: 36101).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 ± 2 mg (39.7 mg/cm2 according to guideline) of the test item was applied onto the surface of the tissue. The tissues were wetted with 25 µL DPBS prior to application.

Duration of treatment / exposure:

60 minutes.

Number of replicates:

Triplicate

Test system

Type of coverage:

other: Topical

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
25 ± 2 mg (39.7 mg/cm2 according to guideline)


NEGATIVE CONTROL
25 ± 2 mg (39.7 mg/cm2)


POSITIVE CONTROL
25 ± 2 mg (39.7 mg/cm2)

Duration of treatment / exposure:

60 minutes

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

freeze killed tissues

Value:

5.59

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

no indication of irritation

Remarks:

Non specific killed control tissues: mean rel. viability 2.19%

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

viable tissues

Value:

7.41

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissues

Value:

108.12

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Pre-Experiment

Assessment of Colour Interference

Treatment Group	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Evaluation Mean OD570 (blank corrected) > 0.08
Blank Aqua Deion.	0.037	0.036	0.037	--	--
Test Item + Aqua Deion.	0.075	0.081	0.078	0.041	no
Blank Isopropanol	0.039	0.038	0.038	--	--
Test Item+ Isopropanol	2.611	2.778	2.694	2.656	yes

The mean OD of the test item in isopropanol was > 0.08 and therefore, an additional test with viable tissues without MTT addition was necessary in the main experiment.

 

Main experiment

Results after treatment with Disperse Red 153 and the controls

 

Dose	Mean OD	Relative	Standard	Mean
Group	of	Viability [%]	Deviation	Relative Viability
	3 Tissues	Tissue 1, 2, 3 ***	[%]	[%]
Negative Control	1.725	1.757	3.9	100.0
Positive Control	0.084	1.733	1.2	4.88
Test Item	1.995	1.700	0.1	108.12
Negative Control Viable Tissues	0.007	0.116	0.3	0.43
Test Item Viable Tissues	0.128	0.111	0.2	7.41
Negative Control Freeze killed Tissues	0.056	0.104	0.0	3.23
Test Item Freeze killed Tissues	0.096	0.225	0.0	5.59
Non Specific Killed Control Tissues	0.038	0.290	0.1	2.19

 

 

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Disperse Red 153 is non-irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the skin irritation potential of Disperse Red 153 by means of the Human Skin Model Test.

The test item proved to be a MTT reducer in the MTT interference pre-experiment. Also, it proved to dye isopropanol in the color interference pre-experiment since the OD of the test item in isopropanol at 570 nm after blank correction was > 0.08. Therefore, additional tests with freeze-killed tissues, viable tissues and NSKC had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the main experiment.

Each three tissues of the human skin model EpiDerm^™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ³ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a decrease in the viability compared with the negative control to 4.88%, thus ensuring the validity of the test system.

The standard deviations between the three tissue percentage viability values of each group (test item, positive and negative controls) in the main test were below 18 (threshold according OECD 439: ≤ 18), thus ensuring the validity of the study.

After treatment with the test item Disperse Red 153 the mean viability value was 108.12% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.