4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17.-18.01.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

EC Number:

287-722-1

EC Name:

4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

Cas Number:

85567-10-8

Molecular formula:

C18H13N4Na3O12S3

IUPAC Name:

trisodium 5-oxo-4-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-1-(4-sulfonatophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylate

Test material form:

solid

Test animals / tissue source

Species:

human

Strain:

other: normal, human-derived epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

- Test system: reconstructed human cornea-line epithelium (RhCE) model, cultured to form a stratified, highly differentiated squamous epithelium morphologically similar
to that found in a human cornea
- Source: EpiOcular™ tissue kits (e.g. OCL-200-EIT; MatTek)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Negative Control 50 μL Aqua dest.
- Positive Control 50 μL methyl acetate
- Test Item 50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2 tissues per dose group

Details on study design:

Experimental Procedure:
- Equilibration: incubated for 1 h in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air in assay medium
- Pre-incubation: in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air for 16 - 24 h
- Pre-treatment: with 20 μL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air
- Treatment (exposure): incubated for 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air
- Post-treatment: incubated for 18 ± 0.25 h at 37 ± 1°C, 5.0% CO2 / 95% air

MTT assay:
Incubation with 0.3 mL of MTT solution for 3 h ± 10 min at 37 ± 1 °C, 5.0% CO2 / 95% air
After incubation with MTT the inserts were incubated with isopropanol and shaken for 2-3 hours at room
temperature.

Data Analysis:
Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities
obtained after treatment compared to the negative control tissues concurrently treated with
Aqua dest.

Test Acceptance Criteria:
The test meets acceptance criteria if:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%.

Deviations:
The deviations did not influence the quality or integrity of the present study.

Results and discussion

In vitro

Results

Irritation parameter:

other: Mean Tissue Viability [%]

Run / experiment:

mean of two tissues

Value:

21.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The controls confirmed the validity of the study.

Any other information on results incl. tables

Result of the Test Item Reactive Yellow 42

Name	Negative control		Positive control		Test item
Tissue	1	2	1	2	1	2
OD570values	1.488	1.419	0.657	0.619	0.232	0.458
	1.513	1.411	0.685	0.622	0.234	0.466
OD570values (blank-corrected)	1.445	1.376	0.613	0.576	0.189	0.415
	1.47	1.368	0.642	0.579	0.191	0.423
Mean of the duplicates	1.457	1.372	0.628	0.577	0.19	0.419
Mean OD	1.414*		0.602		0.304
TOTTT	-		-		0.299
Mean sd OD	0.06		0.04		0.16
Tissue Viability (%)	103	97	44.4	40.8	13.4	29.6
Relative tissue viability difference (%)***	6		3.6		16.2
Mean tissue viability (%)	100		42.6**		21.5

*Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

**Mean relative tissue viability of the positive control is < 50%

***Relative tissue viability difference of replicate tissues is < 20%

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed irritant effects.

According to the OECD Guideline 492, the EpiOcular Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1).
For these purposes, further testing with other suitable test methods may be required.

Executive summary:

The potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. The study was performed in accordance to OECD TG 492 and in compliance to GLP.

In the present study Reactive Yellow 42 was applied topically to the EpiOcular tissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After a 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay.

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest.

The mixture of 50 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.458). The mean relative tissue viability (% negative control) of the positive control was < 50% (42.6%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (16.2%).

The mixture of 50 mg test item per 1 mL A. dest. showed colouring as compared to the solvent.

Since the mean relative tissue viability of the test item treated tissues (TM) was below the 60% threshold value no coloured tissue controls were performed, since the test item has to be classified as irritant in any case.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (21.5%).

In this study under the given conditions the test item showed irritant effects. The test item is classified as “irritant“ in accordance with UN GHS “Category 1” or “Category 2”.

According to the OECD Guideline 492, the EpiOcular Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods may be required.