N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 June 2016 until 18 July 2016

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Test material information

Constituent 1

Reference substance name:

Pigment Additiv FGR

IUPAC Name:

Pigment Additiv FGR

Constituent 2

Reference substance name:

N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

EC Number:

225-691-8

EC Name:

N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

Cas Number:

5012-29-3

Molecular formula:

C25H18Cl3N3O3

IUPAC Name:

N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)diazenyl]-2-naphthamide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: Pigment Additiv FGR
Batch: FB3212.013
CAS-No.: 5012-29-3
EINECS / EC-No.: 225-691-8
Purity: 94.51% (w/w), dose calculation was not be adjusted to purity
Appearance: Brown powder
Expiry Date: 10.05.2020 (Statement of producer)
Storage Conditions: At room temperature
Stability in Solvent: > 72 h in DMSO at room temperature
> 72 h in 1,2-propylene glycol at room temperature
Certificate of Analysis: AZ 577/Toxd2, dated 11. December 2015

In vitro test system

Test system:

human skin model

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item

Duration of treatment / exposure:

60 minutes.

Number of replicates:

3 tissues

Test animals

Species:

other: reconstituted human epidermis model

Details on test animals or test system and environmental conditions:

Identification: Pigment-Additiv FGR
Other name: N-(4-Ethoxylphenyl)-3-hydroxy-4-[(2,4,5-trichloro-phenyl) azo]naphthalene-2-carboxamide
Batch: FB3212.013
EINECS No.: 225-691-8
CAS No: 5012-29-3
Purity: 94.51% (w/w), dose calculation was not adjusted to purity
Appearance: Red-brown powder
Expiry Date: 10 May 2020 (statement of producer)
Storage Conditions: At room temperature
Certificate of Analysis AZ 577/Toxd2, dated 11. December 2015

Test system

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
30 µL DPBS (MatTek) were used as negative control per tissue


POSITIVE CONTROL
- Amount(s) applied (volume or weight):
30 µL of a 5% SLS solution in deionised water (MatTek) were used a positive control per tissue.

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

This value is above the threshold for irritancy of ≤ 50% compared to result of the negg.control.

Run / experiment:

After treatment with the test item Pigment-Additiv FGR the mean relative absorbance value decreased to 96.5% compared to the relative absorbance value of the negative control.

Value:

>= 0.8 - <= 2.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

In vivo

Irritant / corrosive response data:

Compared to the relative absorbance value of the negative control the corrected mean relative absorbance value achieved 92.4% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Other effects:

No

Any other information on results incl. tables

Results after treatment with Pigment-Additiv FGR (60 minutes exposure period):

Dose Group	Tissue No	Absorb. 570 nm Well 1	Absorb. 570 nm Well 2	Absorb. 570 nm Well 3	Mean Absorb. of 3 Wells	Mean Absorb. of 3 Wells corrected	Mean Absorb. of 3 tissues after blank corr.*	Rel. Absorb. [%] Tissue 1, 2 + 3 **	Relative Standard Deviation	Mean Rel. Absorb. [% of Negative Control] ***
Blank		0.038	0.047	0.037	0.041	0.000
Negative Control	1	1.972	1.935	1.892	1.933	1.892	1.810	104.5	4.9	100
	2	1.881	1.831	1.874	1.862	1.821		100.6
	3	1.788	1.738	1.746	1.757	1.717		94.8
Test Item	1	1.703	1.667	1.723	1.698	1.657	1.756	91.5	5.6	97.0
	2	1.887	1.900	1.892	1.893	1.853		102.3
	3	1.803	1.768	1.892	1.798	1.758		97.1
Positive Control	1	0.093	0.091	0.091	0.092	0.051	0.050	2.8	7.6	2.8
	2	0.093	0.094	0.094	0.093	0.053		2.9
	3	0.087	0.086	0.086	0.086	0.046		2.5

Dose Group	Tissue No.	Absorb. 570 nm Well 1	Absorb. 570 nm Well 2	Absorb. 570 nm Well 3	Mean Absorb. of 3 Wells	Mean Absorb. of 3 Wells blank corr.	Rel. Absorb. [%]**	OD ***	Corr. Mean Rel. Absorb. [% of Neg. Contr.] ****
Blank		0.038	0.047	0.037	0.041	0.000		1.747	96.5
Negative Control	1	0.038	0.038	0.039	0.038	-0.002	100.0
Test Item	1	0.049	0.050	0.051	0.0505	0.0090	-432.3

*   Mean of three replicate wells after blank correction

**  relative absorbance per tissue [rounded values]:

*** OD=OD_{coloured tissue(MTT assay)}– OD_{coloured tissue (no MTT assay)}

****relative absorbance per treatment group [rounded values]

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water led to a change in colour. An additional test with one viable tissue was necessary. The result was used for data correction of the results in the main experiment.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. An additional test with freeze-killed tissues was not necessary.

The mean relative absorbance value of the test item after correction, corresponding to the cell viability, was reduced to 96.5% (threshold for irritancy: ≤ 50%) compared to the result of the negative control, consequently the test item was not irritant to skin.

Applicant's summary and conclusion

Interpretation of results:

other: Not irritant

Conclusions:

Compared to the relative absorbance value of the negative control the corrected mean relative absorbance value was reduced to 96.5% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Executive summary:

This in vitro study was performed to assess the irritation potential of Pigment-Additiv FGR by means of the Human Skin Model Test.

The test item passed the MTT interference pre-test. Due to its intensive colour, an additional test with one viable tissue (without MTT addition) was necessary to correct the result in the main experiment.

Approximately 25 mg of the test item and each 30 µL of the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 43.3 hours the tissues were treated with the MTT solution for 3 hours following nearly 69 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

The acceptance criteria were met:

·        Tissue viability was meeting the acceptance criterion if the mean OD₅₇₀of the negative control tissues was³0.8 and≤2.8 (values between 1.738 and 1.972).

·        The mean relative tissue viability of the positive control was£20% (2.8).

·        The relative standard deviations between the % variability values of the test item and the controls in the main test were below 8% (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative controlthe corrected mean relative absorbance value was reduced to 96.5% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.