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

Endpoint summary

Description of key information

The substance has been tested for skin and eye irritation using in vitro methods.

In an in vitro skin irritation test conducted in accordance with OECD Guideline 439, the mean cell/tissue viability was reported as 95.3%.  This indicates that the substance is not a skin irritant under conditions of the test.

In an in vitro eye irritation test conducted in accordance with OECD Guideline 492, the mean cell/tissue viability was reported 91.1%.  This indicates that the substance is not an eye irritant under conditions of the test.

 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

June 18, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended in the test guideline.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: LabCyte EPI-MODEL24 - SIT
- Tissue batch number(s): LCE24-200727-A
- Expiry date: 31 July 2020
- Date of initiation of testing: 29 July 2020

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsed 15 times or more with PBS
- Observable damage in the tissue due to washing: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.5mg/mL per well
- Incubation time: 180 +/- 5 minutes
- Spectrophotometer: FLUOstar Omega, BMG LABTECH
- Wavelength: 570 and 650nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 1.1
- Barrier function: IC50 = 0.27%
- Morphology: Observation of multilayered epidermis with a stratum corneum


NUMBER OF REPLICATE TISSUES: 3

Control samples:

yes, concurrent negative control

Amount/concentration applied:

Negative control: Distilled water - 25µl
Positive control: 5% w/v SDS solution - 25µl
Test item: neat (triplicate) - 25 mg

Duration of treatment / exposure:

15 minutes +/- 3 minutes

Duration of post-treatment incubation (if applicable):

42 +/- 1 hr

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

92.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

90.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

103.4

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
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes - mean measurement OD was 0.737
- Acceptance criteria met for positive control: yes - mean cell viability was 2.8
- Acceptance criteria met for variability between replicate measurements: yes - SDs of cell viability were, 2.7%, 1.0% and 7.1%

Table 2: Result of skin irritation test

Group	Tissue no.	Measurement value		Cell viability^a)(%)		SD^c) (%)	Category
			Mean		Mean^d)
Negative control (Distilled water)	1	0.759	0.737	103.0	100.0	2.7	-
	2	0.721		97.8
	3	0.731		99.2
Positive control (5% w/w SDS solution)	1	0.014	0.021	1.9	2.8	1.0	-
	2	0.02		2.7
	3	0.029		3.9
Test Item^d)	1	0.679	0.702	92.1	95.3	7.1	Non-irritant
	2	0.665		90.2
	3	0.762		103.4

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

^{b) The mean cell viability was calculated from mean OD value of each group}

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

^{d) The mean OD value was not corrected because the staining ratio was <5%}

 

Table 3: Results of tissue-binding test

Group	Tissue no.	Measurement value		Staining ratio^b) (%)
			Mean
Negative contol item^a)	1	0.000	0.0	-
	2	0.000
Test item^a)	1	0.000	0.0	0.0
	2	0.000

^{a) Medium without MTT was used instead of MTT medium}

^{b) staining ratio ([mean OD value of the test item (without MTT)] - [mean OD value of negative control (without MTT)] / [mean OD value of negative control (with MTT)]) x 100}

Interpretation of results:

GHS criteria not met

Conclusions:

The mean cell viability of the test item was 95.3% and therefore the substance is not classified as a skin irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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 

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

Irritation parameter:

mean percent tissue viability 

Value:

91.1

Remarks on result:

no indication of irritation

Irritation parameter:

percent tissue viability 

Run / experiment:

1

Value:

95.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent tissue viability 

Run / experiment:

2

Value:

95.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent tissue viability 

Run / experiment:

3

Value:

81.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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}

 

 

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the in vitro studies conducted on skin and eye, the cell/tissue viabilities when the substance was tested were greater than the values which warrant classification. The substance does not fulfil the criteria of classification as a skin or eye irritant under the CLP regulation (EC1272/2008, as amended).