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

Endpoint summary

Description of key information

The substance is not a skin or eye irritant.

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:

2021-09-23 - 2021-10-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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 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

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

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%

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

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

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

 

 

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

The test was performed on 30 August 2021

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 Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: • Commission Regulation (EU) No 2017/735 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 (Reach).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Freshly isolated bovine cornea (14 month old donor cattle)
Source: AB Schlachthof GmbH, 63739 Aschaffenburg, Germany

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test Item:
The test item was tested as a 20% suspension (w/v) in saline using sonication for 10 minutes.

Negative Controls:
Negative Control 1: Saline (0.9% NaCl in deionised water)
Negative Control 2: Deionized water

Positive Controls:
Positive Control 1: 10% (w/v) Benzalkonium chloride (purity not indicated by the producer) in saline using sonication.
Positive Control 2: 20 % (w/v) Imidazole in saline

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 corneae per group (test item, negative controls, positive controls)

Details on study design:

Three corneas were exposed to each 0.75 mL of a 20% (w/v) suspension of the stest item in physiological saline for 240 minutes.
After treatment the test item suspension was rinsed off the corneas and the corneas' opacity was determined. In a second step the permeability of the corneas was determined photometrically after 90 +/- 5 minutes treatment with fluorescein solution.

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneas and displays a numerical opacity value. This value was recorded in a table. The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Permeability Determination
Following to the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)


Criteria for Determination of a Valid Test

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

A single testing run composed of at least three corneas should be sufficient for a test chemical when the resulting classification is unequivocal. In cases of borderline results in the first testing run, a second testing run will be considered, as well as a third one in case of discordant mean IVIS results between the first two testing runs. A result in the first testing run is considered borderline if the predictions from the 3 corneas are non-concordant, such that:
• 2 of the 3 corneas give discordant predictions from the mean of all corneas, or,
• 1 of the 3 corneas gives a discordant prediction from the mean of all 3 corneas,
and the discordant result is >10 IVIS units from the cut-off threshold of 55.
• If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean IVIS value), then a final decision can be taken without further testing. If the repeat testing run results in a non-concordant prediction from the initial testing run (based upon the mean IVIS value), then a third and final testing run should be conducted to resolve equivocal predictions, and to classify the test chemical. It may be permissible to waive further testing for classification and labelling in the event any testing run results in a GHS Category 1 prediction.

Irritation parameter:

in vitro irritation score

Run / experiment:

main experiment

Value:

12.99

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: prediction for the damage hazard cannot be made (GHS)

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative controls: fulfilled, no category (IVIS 1.42)
- Acceptance criteria met for positive controls: filfilled, category 1 (IVIS 90.02 and 95.97)

Results after 240 Minutes Treatment Time

Test Group	Opacity value #		Permeability #		IVIS	Mean IVIS	Standard deviation IVIS	Proposed IVIS
		Mean		Mean
	0		0.071		1.07
Neg. Control 1	0	0.33	0.065	0.072	0.98	1.42	0.69	No Category
	1		0.081		2.22
	1		0.070		2.05
Neg. Control 2	0		0.073		1.10	1.42	0.54	No category
	0		0.075		1.13
	90.67*		0.225*		94.04
Pos. Control 1	82.67*		0.274*		86.77	90.02	3.69	Category 1
	84.67*		0.306*		89.25
	82.67*		0.942*		90.79
Pos. Control 2	90.67*		0.610*		99.81	95.67	4.32	Category 1
	77.67*		0.909*		91.30
	5.67*		0.446*		12.35
Disperse Red 153	6.67*		0.345*		11.84	12.99	1.57	No prediction
	6.67*		0.541*		14.78

Neg. Control 1 = Saline

Neg. Control 2 = deionised water

Pos. Control 1 = 10% Benzalkonium chloride

Pos. Control 2 = 20% Imidazole

# Opacity calculated as Difference of t₂₄₀ - t₀

## Permeability at 490 nm (OD₄₉₀)

*corrected values

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, a prediction for the damage hazard cannot be made (GHS) for Disperse Red 153.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Disperse Red 153 by means of the BCOP (OECD 437) assay using fresh bovine corneas.

After a first opacity measurement of the fresh bovine corneas (t0), the 20% (w/v) suspensionin saline of the test item Disperse Red 153, the positive, and the negative controls were applied to the different corneas and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneas andopacity was measured again (t240).

After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative controls(saline and deionised water), neither an increase of opacity nor permeability of the corneas could be observed (saline meanIVIS= 1.42 and deionised water mean IVIS = 1.42).

Both positive controls 10% (w/v) Benzalkonium chloride in saline and imidazole showed clear opacity and distinctive permeability of the corneas (mean IVIS = 90.02 and 95.97, respectively) corresponding to a classification as serious eye damaging (EU CLP/GHS (Category 1).

Relative to the negative control, the test item Disperse Red 153 caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 12.99 (threshold for serious eye damage: IVIS > 55). According to OECD 437, no prediction for the damage hazard of the test item to the eye can be made.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An 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.

 

An in vitro study was performed to assess the corneal irritation and damage potential of Disperse Red 153 by means of the BCOP (OECD 437) assay using fresh bovine corneas. After a first opacity measurement of the fresh bovine corneas (t0), the 20% (w/v) suspensionin saline of the test item Disperse Red 153, the positive, and the negative controls were applied to the different corneas and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneas andopacity was measured again (t240). After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative controls(saline and deionised water), neither an increase of opacity nor permeability of the corneas could be observed (saline meanIVIS= 1.42 and deionised water mean IVIS = 1.42). Both positive controls 10% (w/v) Benzalkonium chloride in saline and imidazole showed clear opacity and distinctive permeability of the corneas (mean IVIS = 90.02 and 95.97, respectively) corresponding to a classification as serious eye damaging (EU CLP/GHS (Category 1).

Relative to the negative control, the test item Disperse Red 153 caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 12.99 (threshold for serious eye damage: IVIS > 55). According to OECD 437, no prediction for the damage hazard of the test item to the eye can be made resulting from this value.

The substance was profiled using the OECD QSAR Toolbox. According to this profiling, the substance does not meet the inclusion criteria for eye irritation as defined by the BfR. In addition it meets several exclusion criteria for eye irritation (water solubility, melting point).

The substance is hence not considered to be irritating to the eye.