N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

June 18, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

EC Number:

876-151-9

Cas Number:

2292123-68-1

Molecular formula:

C27H24N2O7S2

IUPAC Name:

N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

RECONSTRUCTED HUMAN CORNEAL (EIT) TISSUE
- Model used: LabCyte CORNEAL-MODEL24 EIT (model recommended in test method)
- Tissue batch number(s): LCC24-200817-A
- Expiry date: 21 August 2020
- Date of initiation of testing: 19 August 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C 

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent no treatment

Amount / concentration applied:

Negative control: No treatment
Positive control: Lauric acid - 10 mg
Test item: neat (triplicate) - 10 mg

Duration of treatment / exposure:

24 hours

Duration of post- treatment incubation (in vitro):

240 minutes +/- 15 minutes

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissue insert is completely submerged three times
(in a beaker with approx. 100mL PBS) for rinsing. Rinsed ten times or more with PBS
- Observable damage in the tissue due to washing: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- WST-8 solution*: 200µL per well
- Incubation time: 240 +/- 15 minutes
- Spectrophotometer: FLUOstar Omega, BMG LABTECH
- Wavelength: 450 and 650nm

*WST-8 solution: Cell counting Kit-8 (WST-8: Lot number NG023, DOJINDO Laboratories) diluted with Earle's balanced salt solution (EBSS: Lot number RNBG9914, SIGMA-ALDRICH). WST-8 : EBSS 1 : 10.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 1.3
- Barrier function: IC50 = 0.28%
- Morphology: Observation of Corneal model(including at least 3 layers of epithelial cells and a non-keratinized surface)

NUMBER OF REPLICATE TISSUES: 3

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

mean percent tissue viability 

Value:

91.1

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

percent tissue viability 

Run / experiment:

1

Value:

95.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

percent tissue viability 

Run / experiment:

2

Value:

95.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

percent tissue viability 

Run / experiment:

3

Value:

81.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 2: Result of eye irritation test

Group	Tissue no.	Measurement value			Cell viability^b), c) (%)		SD^e) ^(%)	Category
			Corrected value^a)	Mean		Mean^d)
Negative control (non-treatment)	1	1.179	-	1.300	90.7	100.0	15.4	-
	2	1.531	-		117.8
	3	1.189	-		91.5
Positive control (Lauric acid)	1	-0.004	-	-0.004	0.0	0.0	0.0	-
	2	-0.005	-		0.0
	3	-0.004	-		0.0
Test Item	1	1.247	1.247	1.184	95.9	91.1	0.0	Non-irritant
	2	1.241	1.241		95.5
	3	1.063	1.063		81.8

^{a) OD value was corrected by the result of the binding test}

^{Corrected measurement value = [OD value of the test item (with WST-8)] - {[mean OD value of othe test item (without WST-8)] / [mean OD value of negative control (without WST-8)]}}

^{b) Cell viability in the negative control was regarded as 100%}

^{c) If cell viability was <0, the value was considered to 0%}

^{d) the mean cell viability was calculated from mean OD value of each group}

^{e) The SD was calculated from the cell viability (n=3) of each tissue insert}

Table 3: Results of tissue-binding test

Group	Tissue no.	Measurement value
			Mean
Negative contol (no treatment)^a)	1	-0.045	-0.045
	2	-0.045
Test item^a)	1	-0.045	-0.045
	2	-0.045

^{a) EBSS was used instead of WST-8 solution}

 

 

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

The mean cell viability of the test item was 91.1% and therefore the substance is not classified as an eye irritant.