Reaction products of 4,4'-Azo-3-hydroxynaphthalene-1-sulphonate coupled with 2-naphthol and 2-amino-4 (or 5)-nitrophenol, chelated with chromium (3+), sodium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20.06. - 22.06.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted: 28 July 2015

Deviations:

yes

Remarks:

The difference of viability between the positive control tissues of was > 18%. Average viability was 28.9% what is < 50%. The highest (outlying) viability is 45.2% what is < 50% as well.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen sodium [3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

EC Number:

267-824-2

EC Name:

Hydrogen sodium [3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

Cas Number:

67939-06-4

Molecular formula:

C36H20CrN5O9SNa

IUPAC Name:

Hydrogen sodium [3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

The reconstructed human cornea-like epithelial model EpiOcular™ (OCL-200 ver. 2.0) comes from MatTek, Bratislava, SK. The RhCE tissues are reconstructed from primary human cells, which have been cultured for several days to form a stratified, highly differentiated squamous epithelium, morphologically similar to that found in the human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance (50 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 20 µL of PBS.

Duration of treatment / exposure:

6h

Duration of post- treatment incubation (in vitro):

after removal:post-soaked-25 min.post-incubation-18 hours

Number of animals or in vitro replicates:

two replicate tissues

Details on study design:

Temperature: 37±1°C3 tissues for the test substance3 tissues for every control2 tissues as colorant controlTest ProcedureOn the day of receipt, EpiOcularTM tissues are conditioned to release transport stress related compounds and debris by incubation in assay medium delivered by MatTek for test performance for 1 hour at standard culture conditions and, after media replacement, overnight (following 16-24 hours) also standard at culture conditions. After pre-incubations, tissues are wetted with 20 μl of PBS spread across entire tissue surface. After 30 minutes incubation tissues are topically exposed to the test chemical (50 mg per tissue) for 6 ± 0.25 hours. Four tissues (2 for MTT test 2 for colorant control) are used per test substance (TS) and two for the positive control (PC) and negative control (NC). At the end of the 6±0.25 hours treatment time, the test articles should be removed by extensively rinsing the tissues with PBS brought to room temperature. After rinsing, tissues are immediately transferred to and immersed in 5 ml of previously warmed assay medium (room temperature) in a pre-labelled 12-well plate for a 25 ± 2 minute immersion incubation (post-soak) at room temperature. This incubation in assay medium is intended to remove any test article absorbed into the tissue. At the end of the post-soak immersion, each insert is removed from the assay medium, the medium is decanted off the tissue, and the insert is blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 ml of warm assay medium. The tissues should be incubated for 18±0.25 hours at standard culture conditions (post-treatment incubation). At the end of the post-treatment incubation, each insert is removed from the 6-well plate and gently blotted on absorbent material. Two tissues are placed into the 24-well plate containing 0.3 ml of MTT solution and incubated for 180±10 minutes at standard culture conditions. Two tissues undergo the same procedure with medium instead MTT solution (colorant control).In the end of staining (incubation with medium) the bottom of all inserts is blotted on absorbent material, and inserts are then transferred to a pre-labelled 6-well plate containing 2 ml of isopropyl alcohol in each well so that no isopropyl alcohol is flowing into the insert. The plates are sealed with parafilm, and are either stored overnight at 2-8°C in the dark or immediately extracted. To extract the MTT, the plates are placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. After extraction extracts are collected, mixed and two 200 μl aliquots are transferred to the appropriate wells of a pre-labeled 96-well plate for OD570 reading.

Results and discussion

In vitro

Results

Irritation parameter:

other: viability of treated tissues

Run / experiment:

1

Value:

-2.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Any other information on results incl. tables

Table No. 1: OD570 values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities.

Treatment	OD570 (tissue 1,2,3)			Avg	SD	%NC
water (NC)	1.906	1.919	1.967	1.931	0.026
viability (%NC)	98.7	99.4	101.9	100.0	1.4	100.0
394/16 (C5)	0.509	0.498	0.644	0.550	0.066
viability (%NC)	26.4	25.8	33.4	28.5	12.0	28.5
394/16 (C5_CC)	0.654	0.551	-	0.603	0.052
viability (%NC)	33.9	28.5	-	31.2	8.6	31.2
99% MA (PC)	0.872	0.442	0.359	0.558	0.225
viability (%NC)	45.2	22.9	18.6	28.9	40.3	28.9

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the above-described experimental design, average viability of tissues treated by the test substance Acid Black 227 was -2.7 % (after correction) of negative control average value. The effect of the test substance was positive in EpiOcularTM model (tissues were damaged). According to the classification criteria given in chapter 4.5., the test substance, Acid Black 227, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Executive summary:

The test substance, Acid Black 227, was assayed for the in vitro eye irritation in human epidermal model EpiOcularTM. The test was performed according to the OECD Test Guideline No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Details of the procedure are given in Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals (MatTek 06/29/2015).

After pre-incubation and wetting of tissues, 50 mg of the test substance was placed directly atop to the tissue and it was spread on the entire tissue surface. Length of exposition was 6 hours at 37±1°C in humidified CO2 incubator (5±1% CO2). Three tissues were used for the test substance and every control. Two tissues more were used as colorant control to correction of possible colour interference, which was undergo the entire testing procedure excepting of incubation with MTT medium.

After removal of the test substance, tissues were post-soaked in medium for approximately 25 minutes and post-incubated for about 18 hours at culture conditions. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Test for colour interference revealed direct reduction caused with the test substance. “Viability” of tissues treated by the test substance was corrected.

Test for direct reduction was performed as a part of another study. Direct reduction was not discernible. With regard to the result of MTT, test assay in frozen tissues was not performed.

Under the above-described experimental design average viability of treated tissues was -2.7 % (after correction) i.e. viability was ≤ 60 %.

The effect of the test substance was positive in EpiOcularTM model (tissues were damaged).

According to the classification criteria given in chapter 4.5., the test substance, Acid Black 227, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1). Further testing with other test methods will be required.