Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-amino-4-hydroxynaphthalene-2-sulphonate]

Endpoint summary

Description of key information

FAT 21021/E is not-irritating to skin and eye.

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:

Experimental starting date - 02 September 2015; Experimental completion date - 07 September 2015; Study completion date - 19 December 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See below

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See below

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 21021/E TE
Batch: 72
Purity: ≥75 %
Physical state/ Appearance: Red solid
Expiry date:16 February 2020
Storage Conditions: room temperature in the dark.

Test system:

human skin model

Remarks:

EPISKIN™ Reconstructed Human Epidermis Model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EPISKIN Reconstructed Human Epidermis Model Kit
- Tissue batch number: 15 EKIN 035
- Date of initiation of testing:
02 September 2015.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
One

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.2 mg/ml
- Incubation time: 3 hours
- Spectrophotometer: UV Vis
- Wavelength: 562nm

NUMBER OF REPLICATE TISSUES: Triplicate

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approximately 10 mg (26.3 mg/cm²) of the test item was applied

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours incubation at 37 °C.

Number of replicates:

Three

Irritation / corrosion parameter:

% tissue viability

Value:

105.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

See "Any other information on results incl. tables"

Direct MTT Reduction

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT. 

 

Assessment of Color Interference with the MTT endpoint

Colored test items may interfere with the MTT endpoint. Therefore, an additional functional procedure using viable color correction tissues was performed to measure any potential interference. However, the results obtained showed that negligible interference due to the color of the test item occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 9.9 % relative to the negative control treated tissues and the standard deviation value of the viability was 1.1 %. The positive control acceptance criterion was therefore satisfied. The mean OD₅₆₂ for the negative control treated tissues was 0.718 and the standard deviation value of the viability was 1.0 %. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.6 %. The test item acceptance criterion was therefore satisfied.

 

Mean OD₅₆₂Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562of tissues	Mean OD562of triplicate tissues	±SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.726	0.718	0.007	101.1	100*	1.0
	0.715			99.6
	0.712			99.2
Positive Control Item	0.064	0.071	0.008	8.9	9.9	1.1
	0.080			11.1
	0.069			9.6
Test Item	0.766	0.755	0.012	106.7	105.1	1.6
	0.755			105.2
	0.743			103.5

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100 %

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant.

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. 

 

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Results

The relative mean viability of the test item treated tissues was 105.1% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

 

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

 

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

EU DSD and CLP Not classified for Irritation.

UN GHS Not classified for Irritation (Category 3 cannot be determined).

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:

Experimental Starting And Completion Date - 24 September 2015; Study Completion Date - 29 October 2015.

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:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: FAT 21021/E TE
Batch: 72
Purity: ≥75 %
Physical state/ Appearance: Red solid
Expiry date: 16 February 2020
Storage Conditions: room temperature in the dark.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Vehicle:

physiological saline

Controls:

yes

Amount / concentration applied:

20 % w/v solution in 0.9 % w/v sodium chloride solution; 0.75 mL.

Duration of treatment / exposure:

240 minutes.

Number of animals or in vitro replicates:

Three corneas were allocated to the test item.

Details on study design:

Test Item Formulation and Experimental Preparation
For the purpose of this study the test item was prepared as a 20 % w/v solution in 0.9 % w/v sodium chloride solution. The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Study Design
Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.


Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.


Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.


Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.


Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.


Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin.

Evaluation of Results
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.


Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.


Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.


In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

Visual Observation
The condition of the cornea was visually assessed post treatment.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3 measurements

Value:

1.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Score 2.4

Positive controls validity:

valid

Remarks:

Score 83.9

Remarks on result:

no indication of irritation

Corneal Epithelium Condition

The corneas treated with the test item or negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

 

Treatment	In Vitro Irritancy Score
Test Item	1.1
Negative Control	2.4
Positive Control	83.9

 

 

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of ≤4.1 and permeability ≤0.105. The negative control acceptance criteria were therefore satisfied.

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	5	3	5	2		0.038
	12	3	5	2		0.017
	14	3	5	2		0.016
				2.0*		0.024♦		2.4
Positive Control	16	2	60	58	56.0	1.469	1.445
	18	3	71	68	66.0	1.615	1.591
	19	3	62	59	57.0	1.839	1.815
					59.7●		1.617●	83.9
Test Item	22	2	5	3	1.0	0.045	0.021
	23	3	7	4	2.0	0.013	0.000
	24	2	4	2	0.0	0.026	0.002
					1.0●		0.008●	1.1

OD= Optical density            

* = Mean of the post-treatment -pre‑treatment values            

♦ = Mean permeability                     

● = Mean corrected value     

Corneal Epithelium Condition Post Treatment

Treatment	Cornea Number	Observation Post Treatment
Negative Control	5	clear
	12	clear
	14	clear
Positive Control	16	cloudy
	18	cloudy
	19	cloudy
Test Item	22	clear
	23	clear
	24	clear

     

Interpretation of results:

GHS criteria not met

Conclusions:

No category. Not requiring classification to UN GHS or EU CLP. The test item is "Not irritating" to eyes.

Executive summary:

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

 

Method

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

 

Interpretation

The test item is classified according to the prediction model below:

 

IVIS	CLASSIFICATION
≤ 3	No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55	No prediction of eye irritation can be made
> 55	Category 1. UN GHS or EU CLP Causes serious eye damage

 

Results

The In Vitro irritancy scores are summarized as follows: 

Treatment	In Vitro Irritancy Score
Test Item	1.1
Negative Control	2.4
Positive Control	83.9

 

Conclusion

No category. Not requiring classification to UN GHS or EU CLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

In a GLP-compliant in vitro skin corrosion study conducted according to OECD Guideline 431 using EPIDERM human skin model, the relative mean viabilities of the test item treated tissues were as follows:

60 minute exposure: 111.0 %

3 minute exposure: 101.8 %

Based on these findings, FAT 21021/E was considered to be not corrosive to the skin. A GLP compliant, OECD guideline 439 followed EPISKINTM reconstructed human epidermis model was used to evaluate the skin irritation potential of the test item FAT 21021 after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The relative mean viability of the test item treated tissues was 105.1 % after the 15 minutes exposure period and 42-hours post-exposure incubation period. Based on the criteria for classification the test item is classified as non-irritant. Based on the data from the key EPISKIN(TM) reconstructed human epidermis model test showing a relative mean viability of the test item treated tissues of 105.1 %, the test substance FAT 21021/E can be regarded as not-irritating to rabbit skin.

 

Eye Irritation/corrosion:

The Bovine Corneal Opacity and Permeability (BCOP) test was performed according to GLP and according to OECD guideline 437. The test item was applied at a concentration of 20 % w/v in 0.9 % w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). After 240 minutes of test substance application, In Vitro Irritancy Score (IVIS) of 38.1 was observed, indicating that no prediction of eye irritation can be made. Based on the data from the key Bovine Corneal Opacity and Permeability (BCOP) test showing In Vitro Irritancy Score (IVIS) of 1.1 which is <55 (criteria for classification), the test substance FAT 21021/E can be regarded as non-irritating to eye.

 

Conclusion:

Taking into consideration data from both skin and eye irritation/corrosion studies, it can be concluded that the test substance FAT 21021/E is non-irritating to skin and eye.

Justification for classification or non-classification

Based on the finding in the skin and eye irritation studies, the test substance does not need to be classified according to Directive 67/548/EEC and according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.