Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-22 to 2017-06-28

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 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)

Version / remarks:

2013-07-26

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010-12-08

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

EC Number:

915-758-6

Molecular formula:

C32H18CrN6O8.Na / C32H22CrN10O8.Na / C32H20CrN8O8.Na

IUPAC Name:

Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: N01-131001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS: Solid / dark brown, pH ca. 5 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study)

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Mannheim, 68165 Mannheim, Germany
- Characteristics of donor animals: > 12 months, < 60 months of age

Test system

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20 % (w/v)

Duration of treatment / exposure:

4 h

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.

QUALITY CHECK OF THE ISOLATED CORNEAS: After the equilibration period, the medium in both chambers was replaced by fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 550 opacity units were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: 750 µL, 4 h

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE:
- Number of washing steps after exposure period: The corneas were incubated in a horizontal position at about 32 °C for approximately 4 hours (non-surfactant solids). The NC and the PC were then removed from the anterior chamber by using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red). Because the test substance could not be removed by using a syringe the epithelium was rinsed with the open chamber method.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
Table 2: Decision criteria of BCOP
IVIS Prediction
< 1.5 No classification for eye irritation*
1.5 – 4.5 Borderline *,***
> 4.5; < 45 No prediction can be made for eye irritation, further testing with another suitable method is required *, **
45 - 65 Borderline *, ***
> 65 Ocular corrosive or severe irritant

* According to OECD Guideline 437 (adopted July 2013), this prediction is possible. However, due to limited accuracy of the BCOP test to correctly identify substances that do not require classification for eye irritation or serious eye damage, this test method should not be the first choice to initiate a bottom-up approach. Based on this statement and the experience of the test facility, test substances not leading to the prediction “ocular corrosive or severe irritant” are generally examined in the EpiOcular test as well.
** The test method according to the OECD Guideline 437 revised and adopted in 2013 does not allow for the evaluation of eye irritation, i. e., the result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study are needed.
*** The “borderline“ evaluation (IVIS 3.0 ± 1.5 and 55.0 ± 10.0) was statistically determined by using historic BASF data and takes the test facility specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

24.2

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 4: Opacity score of the test substance, NC and PC

	Cornea No	Initial Opacity	Final Opacity	Opacity Change	Corrected Opacity Change	Mean	SD
Test substance	7	4.1	38.4	34.3	23.8	24.0	6.8
	8	5.3	46.9	41.6	31.0
	9	5.0	32.9	27.9	17.3
NC	1	5.7	16.4	10.7	NA	10.6	4.1
	2	3.6	18.2	14.6	NA
	3	1.7	8.1	6.4	NA
PC	4	4.4	106.9	102.5	91.9	84.5	14.5
	5	4.2	108.6	104.3	93.8
	6	4.6	83.0	78.4	67.9

Table 5: Permeability score of the test substance, NC and PC

	Cornea No	Mean OD490	Dilution Factor	Mean Corrected OD490*	Mean	SD
Test substance	7	0.010	1	0.007	0.011	0.004
	8	0.014	1	0.011
	9	0.018	1	0.016
NC	1	0.000	1	NA	0.003	0.002
	2	0.002	1	NA
	3	0.005	1	NA
PC	4	0.348	5	1.737	2.259	0.454
	5	0.495	5	2.472
	6	0.514	5	2.567

Table 6: In Vitro Irritancy score (IVIS) of the test substance, NC and PC

	Cornea No	Opacity per cornea	Permeability per cornea	IVIS
				Per cornea	Per group
					Mean	SD
Test substance	7	23.8	0.007	23.9	24.2	6.8
	8	31.0	0.011	31.2
	9	17.3	0016	17.6
NC	1	10.7	0.000	10.7	10.6	4.1
	2	14.6	0.002	14.6
	3	6.4	0.005	6.4
PC	4	91.9	1.737	118.0	118.4	12.3
	5	93.8	2.472	130.9
	6	67.9	2.567	106.4

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met