Reaction mass of isomers 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt and 4-[[2-[(Aminocarbonyl)amino]-4-[(4,6-difluoro-2-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-04-09 to 2013-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants), adopted: 7 September 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und dLebensmittelsicherheit, München, Germany)

Test material

Test material information

Constituent 1

Reference substance name:

Reactive Golden Yellow HF-RN 1331

IUPAC Name:

Reactive Golden Yellow HF-RN 1331

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: Reactive Golden Yellow HF-RN 1331
Batch No.: Kilo 10&12
Chemical Name: 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4 pyrimidinyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt
Formula: C17H11F2N7Na2O7S2
Physical State: solid (powder)
Colour: reddish orange
pH: 4.5 at 5%
Molecular Weight: 573.42 g/mol
Purity: 100 % (composition see AZ. No. 12-1327/2473 v.2, dated 2012-10-08)
Storage Conditions: room temperature
Expiry Date: 17.01.2014
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test system

Vehicle:

physiological saline

Amount / concentration applied:

The test item was diluted with physiological saline 0.9% NaCl to gain a 20% concentration.

Duration of treatment / exposure:

750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed and the epithelium washed and an opacity measurement was performed.

Details on study design:

Test System

Preparation of the Corneas:
The assay uses isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals (from Attenberger Fleisch GmbH & Co. KG).
On the test day, fresh eyes were collected from the slautherhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (MC2, Clermont, France) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior
chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +/- 1 °C in a water bath.

Calibration of the Opacitometer:
The opacitometer had been switched on 15 min before the calibration procedure was started. Empty cornea holders were placed into the opacitometer and the readout was adjusted to zero using the “BAL”-turning knob. For calibration the polyester foil no. 1 was introduced into the test chamber
and the readout was adjusted to 75 using the “CAL”-turning knob. To test the linearity of the measurement, two additional calibration foils, polyester foil no. 2 and polyester foil no. 3, were measured. For these, the opacitometer was supposed to display 150 and 225, respectively (± 3%). If this had not been the case, the calibration procedure would have had to be repeated. The calibration procedure was performed before each test and was
documented in the raw data.

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh Complete RPMI. An initial opacity measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France). Three corneas with opacity readings approximately
equivalent to the median opacity of all corneas were selected as negative-control corneas. The opacity of each cornea was read against an air-filled
chamber and recorded. Corneas that have an initial opacity reading above 7 units were not dosed. The medium was removed from the anterior
chamber and replaced with the test item or control.
750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed and the epithelium washed
at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes
at 32 +/- 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined,
using a spectrophotometer.

Test Groups:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl
The BCOP assay is considered to be valid if the in vitro score obtained with the positive control falls within the two standard deviations of the current historical mean.

Evaluation of Results:
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were
corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each
treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank wells were calculated. The mean blank OD490 was subtracted from the OD490 of each well (corrected OD490).
Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were
taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive
control were calculated by subtracting the average corrected OD490 of the negative control corneas from the corrected OD490 value of each
treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas
for that treatment condition.
The following formula was used to determine the in vitro score:
In vitro score = mean opacity value + (15 x mean OD490 value)

Results and discussion

In vivo

Results

Irritation parameter:

other: in vitro irritation score (IVIS)

Basis:

mean

Score:

16.99

Irritant / corrosive response data:

The eye irritancy potential of Reactive Golden Yellow HF-RN 1331 was investigated in the bovine corneal opacity and permeability assay.
The test item was suspended with physiological saline 0.9% NaCl to gain a 20% concentration.
The following mean in vitro irritation score was calculated:
16.99
Therefore the test item was considered as mild irritant.
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

Any other information on results incl. tables

Opacitiy

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative	4	4	0
2	Control	4	5	1
3		4	5	1
MV		4.00	4.67	0.67
4	Positive	6	145	139	138.33
5	Control	6	203	197	196.33
6		5	200	195	194.33
MV		5.67	182.67	177.00	176.33
7	Test Item	5	19	14	13.33
8		3	16	13	12.33
9		2	20	18	17.33
MV		3.33	18.33	15.00	14.33

Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative	0.017
2	Control	0.014
3		0.009
MV		0.013
4	Positive	2.086	2.073
5	Control	2.158	2.145
6		2.118	2.105
MV		2.121	2.107
7	Test Item	0.215	0.202
8		0.156	0.143
9		0.200	0.187
MV		0.190	0.177

In vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity Value	Corrected OD490 Value	IVIS
1	Negative	0.00	0.017
2	Control	1.00	0.014
3		1.00	0.009
MV		0.67	0.013	0.87
4	Positive	138.33	2.073
5	Control	196.33	2.145
6		194.33	2.105
MV		176.33	2.107	207.94
7	Test Item	13.33	0.202
8		12.33	0.143
9		17.33	0.187
MV		14.33	0.177	16.99

Applicant's summary and conclusion

Conclusions:

Based on the results of this in vitro study and under the given conditions the test item Reactive Yellow HF-RN 1331 is considered to be mild irritant
to the eye.

Executive summary:

Summary Results

The eye irritancypotential of Reactive Golden Yellow HF-RN 1331 was investigated in the bovine corneal opacity and permeability assay.

Preparation of the test item:                     suspended with physiological saline 0.9% NaCl to gain a 20% concentration

Meanin vitroirritation score:                  16.99

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

Conclusion

Based on the results of this in vitro study and under the given conditions the test item Reactive Yellow HF-RN 1331 is considered to be mild irritant to the eye.