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

Endpoint summary

Description of key information

In an in vitro skin irritation assay 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.

According to the available BCOP study, Pigment Additiv FGR does not require classification for eye irritation or serious eye damage.

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:

07 June 2016 until 18 July 2016

Reliability:

1 (reliable without restriction)

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)

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 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

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

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

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

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

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.

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.

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 26 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

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:

other: OECD Guideline for Testing of Chemicals 437: Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (July, 2013)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 25 April 2012, 23 ,25,Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Vehicle:

physiological saline

Amount / concentration applied:

The test item was tested as a 20% suspension (w/v) in saline. The test item was crushed in mortar with a pistil to improve its consistency. The test item could not be suspended or solved homogeneously, therefore, each 0.75 mL of the prepared stock was distributed to each cornea.

Amount(s) applied (volume or weight with unit): 0.75 mL

Duration of treatment / exposure:

240 minutes

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 minutes treatment with fluorescein solution.


SCORING SYSTEM:

Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae, 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 corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the test groups and after rinsing the opacity value was determined again (t240).


Permeability Determination
Following to the opacity readings, the permeability 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 the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
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:
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 mean IVIS obtained, the test item is classified according to OECD guideline 437 as follows:

IVIS: UN GHS

≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 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.

Irritation parameter:

in vitro irritation score

Value:

1.37

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Relative to the negative control, the test item Pigment Additiv FGR did not cause an increase of the corneal opacity or permeability.

Other effects:

The calculated mean IVIS was 1.37 (Decission Criteria ≤ 3: no categorie, > 3; ≤ 55: no prediction can be made, > 55: Category 1). Based on these findings and according to OECD 437 the test item does not require classification for eye irritation or serious eye damage (UN GHS No Category).

Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Classification UN GHS
		Mean		Mean
Negative Control	0	0	0.080	0.079	1.20	1.19	No Category
	0		0.075		1.13
	0		0.082		1.23
Positive Control	140.00*		0.016*		140.24	128.59	Category 1
	130.00*		0.038*		130.57
	115.00*		-0.003*		114.96
Pigment Additiv FGR	3.00*		0.025*		3.38	1.37	No Category
	1.00*		0.001*		1.02
	0.00*		-0.018*		-0.27

*corrected values

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, Pigment Additiv FGR does not require classification for eye irritation or serious eye damage (UN GHS No Category).

Executive summary:

This in vitro study was performed to assess the corneal damage potential of Pigment Additiv FGR by means of the BCOP assay using fresh bovine corneae.

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

After the opacity measurements permeability of the corneae 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 control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS =1.19).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =128.59) corresponding to a classification asserious eye damaging(CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test itemPigment Additiv FGRdid not cause an increaseof the corneal opacityor permeability.The calculated mean IVIS was1.37(Decission Criteria ≤ 3: no categorie, > 3; ≤ 55: no prediction can be made, > 55: Category 1). Based on these findings and according to OECD 437 the test itemdoes not require classification for eye irritation or serious eye damage (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification

Neither skin irrittion potential nor eye irritation potential was detected in relevant studies.