N-phenyl-N'-[(phenylamino)sulfonyl]urea

Endpoint summary

Description of key information

Guideline studies available for skin and eye irritation

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

Study period:

25. February - 03. May 2016

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)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult human-derived epidermal keratinocytes

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

The EPISKINTM–(SM) model has been validated for irritation testing in an international validation study [9] and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
EPISKINTM –(SM) (Manufacturer: SkinEthic, France, Batch No.:16-EKIN-009, Expiry Date: 07 March 2016)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 23.1-25.7°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the 15 minutes incubation time, the EPISKINTM –(SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
After the 42 hours incubation, all EPISKINTM –(SM) units (except of two colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM –(SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function:
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES:
In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Approximately 10 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded:
-Test items which do not react with MTT: yellow
-Test items reacting with MTT: blue or purple
After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test items did not react with MTT and therefore the use of additional controls was not necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
one

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
according to TG 439:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg of the test item

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 of 1

Value:

98.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the three negative control tissues was in the recommended range (0.858). Standard deviation of the viability results for negative control samples was 7.6.
The positive control treated tissues showed 4.2 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.1.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 7.9.
The mean OD value of the blank samples (acidified isopropanol) was 0.049.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this in vitro EPISKIN model test with N-phenyl-N'[(phenylamino)sulfonyl]urea (Batch number: 150204), the results indicate that the test item is non-irritant to skin.

Executive summary:

An in vitro skin irritation test of N-phenyl-N'[(phenylamino)sulfonyl]urea was performed in a reconstructed human epidermis model. EPISKINTM –(SM)is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The irritation potential of the test item was evaluated according to the OECD No. 439 guideline [1].

Disks of EPISKINTM –(SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control, the test item is considered to be irritant to skin.

Following exposure with N-phenyl-N'[(phenylamino)sulfonyl]urea, the mean cell viability was 98.8 % compared to the negative control. This is above the threshold of 50 %, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

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

Study period:

30. March - 26. May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch/Lot number: 150204
Appearance: White powder
Purity (HPLC): 99.68%
Manufacture date: 09 February 2015
Expiry date: 30 June 2016
Storage conditions: Controlled room temperature (15-25°C, below 70 RH%)

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

TARAVIS Kft. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg

Duration of treatment / exposure:

10 s

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

no post treatment incubation

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2 % (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5 % within the -45 min and the zero time. In each experiment no changes in corneal thickness were observed. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Name: Physiological saline (Salsol solution, 0.9 % (w/v) NaCl)
Batch number: 52532Y05-2
Manufacturer: B. Braun Pharmaceuticals SA
Expiry date: 31 May 2018
Storage condition: Room temperature

SOLVENT CONTROL USED
not applicable

POSITIVE CONTROL USED
Test Item: Imidazole
CAS Number: 288-32-4
Manufacturer: Sigma-Aldrich Co.
Batch number: SLBH7184V
Expiry date: 30 September 2016
Storage conditions: Room temperature
APPLICATION DOSE AND EXPOSURE TIME

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test item if possible.

METHODS FOR MEASURED ENDPOINTS:
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 900 slit-lamp microscope was used for the measurements.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: decision criteria as indicated in the TG was used.

Irritation parameter:

cornea opacity score

Run / experiment:

1st

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

1st experiment

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

1st experiment at 240 min

Value:

< 0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

2nd experiment

Value:

0.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

2nd experiment

Value:

0.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

2nd experiment at 240 min

Value:

1.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

In each experiment, the results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in each experiment. This study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these in vitro eye irritation assays in isolated chicken eyes with N-phenyl-N'[(phenylamino)sulfonyl]urea, the test item was non-irritant, UN GHS Classification: No Category.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (26 July 2013).

After the zero reference measurements, the eye was held in horizontal position and 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the experiments were considered to be valid.

As the test item was solid, the observed negative result of the first experiment was confirmed by a second experiment.

Experiment I: No corneal swelling (<= 5 %) was observed during the four-hour observation period on test item treated eyes. Very slight or slight corneal opacity change (severity 0.5 or 1) was noted on two eyes. No fluorescein retention change was observed on the test item treated eyes. No other corneal effect was observed.

Experiment II:

No corneal swelling (<= 5 %) was observed during the four

-hour observation period on test item treated eyes. Very slight corneal opacity change (severity 0.5) was noted on one eye. Very slight fluorescein retention change change (severity 0.5) was noted on one eye. No other corneal effect was observed.

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

The available information is conclusive but not sufficient for classification.