Acid Yellow 25:1

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10.08.-11.08.2016

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:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

Adopted 26th July 2013.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-(3-methyl-4-(4-methyl-3-(phenylaminosulphonyl)phenylazo)-5-hydroxypyrazol-1-yl)benzenesulphonate

EC Number:

228-810-1

EC Name:

Sodium 4-(3-methyl-4-(4-methyl-3-(phenylaminosulphonyl)phenylazo)-5-hydroxypyrazol-1-yl)benzenesulphonate

Cas Number:

6359-85-9

Molecular formula:

C23H21N5O6S2.Na

IUPAC Name:

sodium 4-(5-hydroxy-3-methyl-4-{[4-methyl-3-(phenylsulfamoyl)phenyl]diazenyl}-1H-pyrazol-1-yl)benzenesulfonate

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

Details on test material:

- Name of test material (as cited in study report): Acid Yellow 25
- Physical state: yellow solid, powder
- Analytical purity: 95 % (w/w)
- Impurities (identity and concentrations): 5%(w/w) NaCl (CAS No. 7647-14-5
- Lot/batch No.: 7003/2007
- Expiration date of the lot/batch: 2 years/June 2018
- Storage condition of test material: The test substance should be stored in dry room in dark in closed container at the room temperature.

Test animals / tissue source

Species:

other: Bovine eye

Details on test animals or tissues and environmental conditions:

Bovine eyes source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic
Eyes were collected by slaughterhouse employees The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 30 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test. The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).

Test system

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance was tested as suspension prepared from test substance at 20% concentration in a 0.9% sodium chloride solution. 2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution.

Duration of treatment / exposure:

4hr

Duration of post- treatment incubation (in vitro):

1,5 hr

Number of animals or in vitro replicates:

Exposed group (test substance) - 3 corneas (No. 7, 10, 13)
Positive control group (20% Imidazole in 0.9% NaCl) – 3 corneas (No. 14, 17, 19)
Negative control group (0.9% NaCl) –3 corneas (No. 2, 9, 12)

Details on study design:

PROCEDURE SCHEME
Selection of corneas, mounting in holders → incubation with EMEM 1hour (32 ± 1°C) → removed EMEM, measurement of baseline opacity → treatment by positive and negative control substances and test substance (incubation 4 hours) → washing of epithelium, measurement of opacity after application → application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C) → measurement of absorbance (490 nm).


SELECTION AND PREPARATION OF CORNEAS
The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects were used.
The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded. From 25 eyes the 9 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 2, 7, 9, 10, 12, 13, 14, 17 and 19), 4 eyes were superfluous and the remaining 3 eyes were used for the testing of another substance.

QUALITY CHECK OF THE ISOLATED CORNEAS
The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).

NEGATIVE CONTROL USED: YES
Name: 0.9% NaCl
Lot: 15IM422A1
Expiration:11/2018
Supplier: Fresenino Kabi s.r.l., Italy


POSITIVE CONTROL USED: YES
Name: Imidazole
Lot no: WXBC1234V
Expiration: 01/2024
Supplier: Sigma-Aldrich

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

0.32

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Table 3: Appearance of corneas  

Group	Cornea No.	Appearance after exposure
Negative control (0.9% NaCl)	2	Without macroscopic damage
	9	Without macroscopic damage
	12	Without macroscopic damage
Positive control (20% Imidazole in 0.9% NaCl)	14	Corneal opacity
	17	Corneal opacity
	19	Corneal opacity
Test substance (Acid Yellow 25)	7	Without macroscopic damage
	10	Without macroscopic damage
	13	Without macroscopic damage

Table 4: Opacity values

Group	Cornea No.	Baseline opacity	Opacity after treatment	Opacity difference	Mean opacity difference	Mean Opacity(corrected)
NC (0.9% NaCl)	2	6	6	0	1.00	-
	9	6	8	2
	12	6	7	1
PC (20% Imidazole in 0.9% NaCl)	14	7	59	52	53.00	(53.00 – 1.00) 52.00
	17	6	62	56
	19	5	56	51
EXP (Acid Yellow 25)	7	4	5	1	1.33	(1.33 – 1.00) 0.33
	10	5	7	2
	13	6	7	1

Note:NC- negative control, PC- positive control, EXP – test substance application form

 

Table 5: Values of permeability (Optical density values)

Group	Cornea No.	Values of Permeability(Optical density  at 490nm)	Mean Permeability	Mean Permeability  (corrected)
NC (0.9% NaCl)	2	0.007	0.011	-
	9	0.018
	12	0.007
PC (20% Imidazole in 0.9% NaCl)	14	2.321	2.015	(2.015 – 0.011)  2.004
	17	1.964
	19	1.761
EXP (Acid Yellow 25)	7	0.001	0.010	(0.010 – 0.011)  - 0.001
	10	0.021
	13	0.008

Table 6: IVIS values

Group	IVIS
	Calculation	Result
NC (0.9% NaCl)	1.00 + 15 x 0.011	1.17
PC (20% Imidazole in 0.9% NaCl)	52.00 + 15 x 2.004	82.06
EXP (Acid Yellow 25)	0.33 + 15 x (- 0.001)	0.32

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The In Vitro Irritancy Score (IVIS) for was 0.32.
This value of IVIS is ≤ 3 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: No category.

Executive summary:

The test substance was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to the OECD Test Guideline No. 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, Adopted 26th July 2013.

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used.

Closed-chamber method was used, because the test substance was applicable by micropipette. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

The In Vitro Irritancy Score (IVIS) for was 0.32.

This value of IVIS is ≤ 3 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: No category.