4-(2-phenylpropan-2-yl)phenyl 3-diazo-4-oxo-3,4-dihydronaphthalene-1-sulfonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 - 28 Feb 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 in 2015

Deviations:

yes

Remarks:

No information on historical data given

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

4-(2-phenylpropan-2-yl)phenyl 4-oxo-3-(λ⁵-diazynylidene)-3,4-dihydronaphthalene-1-sulfonate

Cas Number:

23121-00-8

Molecular formula:

C25H20N2O4S

IUPAC Name:

4-(2-phenylpropan-2-yl)phenyl 4-oxo-3-(λ⁵-diazynylidene)-3,4-dihydronaphthalene-1-sulfonate

Test material form:

solid

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell
The EpiOcular™ human cell construct (OCL-200, OCL-212, MatTek Corporation) is used in this assay. It is a nonkeratinized epithelium prepared from normal human keratinocytes. All biological components of the EpiOcular™ tissue and the kit culture medium have been tested and are free of contamination. Analyses for tissue functionality and quality (like barrier function, viability, morphology were performed and accepted.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 mg
- Concentration: neat/ undiluted

Duration of treatment / exposure:

6 h ± 15 min

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

25 ± 2 min (Post-exposure immersion incubation)
18 h ± 15 min (Post-treatment incubation)

Number of animals or in vitro replicates:

2 per test chemical and controls

Details on study design:

- Details of the test procedure used:
The cytotoxicity of the test item (and thus the ocular irritation potential) is evaluated by the relative viability of the treated RhCE tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT in control and test item treated cultures.

- RhCE tissue construct used, including batch number:
The EpiOcularTM human cell construct (OCL-200 and OCL-212, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot number: 23769)

- Doses of test chemical and control substances used:
50 mg test item; 50 μL positive control (methyl acetate) and 50 μL negative control (sterile deionized water)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
6 h ± 15 min (Treatment/ exposure) at 37°C and 5% CO2
25 ± 2 min (Post-exposure immersion incubation) at room temperature
18 h ± 15 min (Post-treatment incubation) at 37°C and 5% CO2

- Wavelength used for quantifying MTT formazan:
570 nm

- Description of the method used to quantify MTT formazan:
Inserts were removed from the 24-well plate after the incubation time in MTT solution (1 mg/mL) (3 h ± 10 min), the bottom of the insert was blotted on absorbent material and transferred to a 6-well plate containing 2 mL isopropanol per well in a manner avoiding the isopropanol to flow into the insert. The plate was sealed with standard plate sealer. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2-3 h at room temperature. 200 μL samples from each tube were placed into the wells of a 96-well plate and the absorbance/optical density was measured in a 96-well plate spectrophotometer to determine cell viability.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
The test item requires classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60% of the negative control.
The test item requires no classification and labelling according to UN GHS (No Category), if the mean percent tissue viability after exposure and post-exposure incubation is > 60%.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
No information is given on historical data, but the results obtained for the negative and positive control are meeting the acceptance criteria given in the OECD guideline 492.

- Complete supporting information for the specific RhCE tissue construct used:
Data of the Analysis for tissue functionality and quality is included in the study report.

- Reference to historical data of the RhCE tissue construct:
no further information available

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals:
no information provided

- Positive and negative control means and acceptance ranges based on historical data/ TG OECD 492:
negative control mean OD: 1.479 (acceptance range: 0.8 - 2.5)
positive control mean viablity: 46.9% (acceptance criteria: < 50% of the negative control)

- Acceptable variability between tissue replicates for positive and negative controls:
The variability between tissue replicates for positive and negative controls were 0.2 and 1.4% respectively, thus meeting the acceptance criteria of < 20%.

- Acceptable variability between tissue replicates for the test chemical
The variability between tissue replicates for the test chemical was 1.4, thus meeting the acceptance criteria of < 20%.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: mean viability (% of negative control)

Run / experiment:

positive control

Value:

46.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: mean viability (% of negative control)

Run / experiment:

test item

Value:

100.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no information provided

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, as the mean OD value (1.490) of the two negative control tissues lies between 0.8 and 2.5.
- Acceptance criteria met for positive control: Yes, as the mean percentage viability for the positive control (46.9%) lies below 50% of the control viability.

Any other information on results incl. tables

Table 1: MTT results of eye irritation study

Controls		Optical Density (OD)	Viability (%)	Δ%
Negative Control: Sterile deionized water	1	1.468	99.3	1.4
	2	1.490	100.7
	mean	1.479	100.0	-
	standard deviation (SD)		0.99
Positive Control: Methyl acetate	1	0.695	47.0	0.2
	2	0.692	46.8
	mean	0.694	46.9	-
	standard deviation (SD)		0.14
Test item	1	1.501	101.5	1.4
	2	1.480	100.1
	mean	1.491	100.8	-
	standard deviation (SD)		0.99	-

 

Applicant's summary and conclusion

Interpretation of results:

other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified