Fenuron

Endpoint summary

Description of key information

Key in vitro studies are available as prescribed by the ECHA Information requirements for skin and eye irritation/corrosion. Skin irritation/corrosion was tested in a bottom-up approach in an EpiDerm™ artificial three-dimensional model for human skin irritation: the mean viability of cells exposed to Fenuron was 99.7%, which is above the limit of 50%. Eye irritation/corrosion was tested in a top-down approach in a Bovine Corneal Opacity and Permeability Assay (BCOP): the in vitro Irritation Score (IVIS) was 0.285, which is below the cut-off value of 3. In conclusion, Fenuron was considered to be non-irritating/non-corrosive to skin and eye.

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:

2018

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:

adopted 28 July 2015

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 06, 2012

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Isochem Kautschuk GmbH, Batch no. 0010416
- Expiration date of the lot/batch: April 2019
- Purity test date: 5 October 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material : at room temperature (+10°C to +25°C) in a tightly closed container in a dry, cool and well-ventilated place, avoiding exposure to sunlight and moisture

OTHER SPECIFICS: IsoQure UR 300

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM (EPI-200, Lot no. 25876) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.

Justification for test system used:

Skin irritation by cytotoxic action of substances with direct human skin contact refers to the production of reversible damage to the skin following the application of a test item for up to 4 hours [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS)]. This test method provides an in vitro procedure that, depending on information requirements, may allow determining the cytotoxic potency and skin irritancy of test items as a stand-alone replacement test within a testing strategy, in a weight of evidence approach.

Vehicle:

other: Dulbecco’s phosphate buffered saline

Remarks:

For better contact of the test item to the skin, the skin surface was moistened .

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200)
- Tissue batch number(s): Lot no. 25876, Keratinocyte strain 00267
- Date of initiation of testing: January 23, 2018 (start experimental phase)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C, 5% CO2 and 95% relative humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.
- Temperature of post-treatment incubation (if applicable): A post-treatment incubation period of the rinsed tissues in 0.9 mL fresh assay medium of 42 hours was performed; no data on temperature.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS).
- Observable damage in the tissue due to washing: Not specified.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: Each skin sample was placed in MTT assay solution of 1 mg/mL for 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Specification: MTT QC assay, 4 hours. n=3; Acceptance criteria: OD (540-570 nm) 1.0-3.0 ; Result: 1.554 ± 0.167.
- Barrier function: Specification: ET-50 assay. 100 µL 1% Triton X-100, 4 time-points. n=3, MTT assay; Acceptance criteria: ET-50 (4.77-8.72 hrs); Result: 6.95 hrs.
- Morphology: Normal human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) were present under a functional stratum corneum. The Stratum corneum was multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of the cytotoxic marker substance sodium dodecyl sulphate (SDS).
- Contamination: Specification sterility: Long term antibiotic and antimycotic free culture; Acceptance criteria: No contamination; Result: Sterile
The cells used to produce EpiDerm™ tissue are screened for potential biological contaminants. Tests for each potential biological contaminant listed below were performed according to the test method given. Results of "Not detected" indicate that testing for the potential biological contaminant was not observed as determined by the stated test method.
HIV-1 virus - Oligonucleotide-directed amplification: Not detected
Hepatitis B virus - Oligonucleotide- directed amplification: Not detected
Hepatitis C virus - Oligonucleotide- directed amplification: Not detected
Bacteria, yeast. and other fungi - long term antibiotic. antimycotic free culture: Not detected

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No direct MYY interference

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 Independent test sequence was performed. The measurements were made for each of the three tissues in 2 replicates.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 60 minutes exposure is less than or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after 60 minutes exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL
For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): D-PBS. 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% aqueous sodium dodecyl sulphate (SDS)

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Details on study design:

TEST SITE
- Area of exposure: skin surface
- % coverage: 0.63 cm2

REMOVAL OF TEST SUBSTANCE
- Washing: washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)
- Time after start of exposure: 60 minutes

SCORING SYSTEM:
- Method of calculation: According to the EU and GHS classification (H314 or H315 / Category 1/2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.
mean tissue viability ≤ 50% Irritant (I), (H314 or H315 or GHS Category 1 or 2 )
mean tissue viability > 50% non-irritant (NI).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item

Value:

99.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 60 min exposure, 42 h post-treatment incubation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative control (D-PBS)

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 60 min exposure, 42 h post-treatment incubation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control (5% SDS )

Value:

7.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 60 min exposure, 42 h post-treatment incubation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: 25 mg IsoQure UR 300 were added to 1 mL MTT solution and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. Untreated MTT solution was used as control. No change of colour was noted.
In conclusion, no possible interference with the MTT measurement (OD 540 nm), due to colour changes or direct interacting with the MTT assay reagent, was noted and no additional test had to be performed.
- Colour interference with MTT: 25 mg test item were mixed with 300 µL sterile deionised water and incubated in the dark at 37 °C, 5% CO2 and 95% relative humidity for 60 minutes. At the end of exposure time, the mixture was evaluated of the presence and intensity of the staining. No discolouration of the test item was noted. In addition, 25 mg test item were mixed with 2 mL isopropanol and incubated at room temperature for three hours. No discolouration of the test item was noted. Hence, the criteria of the test on colour interference were met.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use.
The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 1.0 and ≤ 2.5. The mean optical density (OD) of 3 negative control tissues was 1.320 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.
- Acceptance criteria met for positive control: A 5% SDS (in H2O) solution was used as a positive control (PC) and tested concurrently with the test chemicals. Concurrent means here that the PC has to be tested in each assay, but only one PC is required per testing day.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as% of the negative control tissues is ≤ 20%. The viability of cells treated with the positive reference item, 5% SDS, was 7.5% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
- Acceptance criteria met for variability between replicate measurements: Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low.
The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual% tissue viabilities of the 3 identically treated replicates is ≤ 18%. The standard deviation determined for all triplicates was below the limit of acceptance of 18%.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions, IsoQure UR 300 tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, predicted to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin. Hence, the test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category).

Executive summary:

A key in vitro study was performed to determine cytotoxic properties of IsoQure UR 300 to skin cells, which might lead to irritation of human skin, by using an artificial three-dimensional model of human skin. The EpiDerm^TM model was employed.

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. IsoQure UR 300 was applied as solid test item to the model skin surface, which was moistened with Dulbecco’s phosphate buffered saline (D-PBS).D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium. The mean viability of cells exposed to IsoQure UR 300 was 99.7% of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. IsoQure UR 300was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of 3 negative control tissues was 1.320 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5. The viability of cells treated with the positive reference item, 5% SDS, was 7.5% of the negative control and fulfilled the acceptance criterion of ≤ 20%. The standard deviation of all triplicates determined was below the limit of acceptance of 18%. Hence, all acceptance criteria were fulfilled.

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:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 09 October 2017

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

February 14, 2017

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Isochem Kautschuk GmbH, Batch no. 0010416
- Expiration date of the lot/batch: April 2019
- Purity test date: 5 October 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material : at room temperature (+10°C to +25°C) in a tightly closed container in a dry, cool and well-ventilated place, avoiding exposure to sunlight and moisture

OTHER SPECIFICS: IsoQure UR 300

Species:

cattle

Strain:

other: Bovine eyes

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, 49699 Lindern, Germany
- Characteristics of donor animals (e.g. age, sex, weight): 6 to 12 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): On collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL .
- Indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL .

Vehicle:

physiological saline

Remarks:

0.9% sodium chloride solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% (w/v)

VEHICLE / NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution):0.9% sodium chloride solution
- Lot/batch no. (if required): Batch no. 173568002; B. Braun Melsungen AG, 34212 Melsungen, Germany

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

Three corneas were used for each treatment group (test item, negative control and positive control).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.
The open-chamber method was used. The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM) , while preventing bubble formation. The corneal holder was equilibrated at 32 °C ± 1 °C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment, solvent and positive control groups.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
0.9% sodium chloride solution

SOLVENT CONTROL USED = NEGATIVE CONTROL = VEHICLE CONTROL

POSITIVE CONTROL USED:
20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
750 µL / 240 minutes

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32 ± 1 °C for 90 ± 5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader (Tecan Sunrise Magellan Version 7.2 ). Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer (Tecan Sunrise) using a standard 1 cm path length.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA:
IVIS UN GHS
≤ 3 No Category
> 3 and ≤ 55 No prediction can be made
> 55 Category 1

Irritation parameter:

in vitro irritation score

Remarks:

mean test substance

Run / experiment:

240 min exposure time

Value:

0.285

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test material

Remarks:

standard deviation ± 0.362

Irritation parameter:

in vitro irritation score

Remarks:

cornea 7

Run / experiment:

240 min exposure time

Value:

0.41

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test material

Irritation parameter:

in vitro irritation score

Remarks:

cornea 8

Run / experiment:

240 min exposure time

Value:

0.569

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test material

Irritation parameter:

in vitro irritation score

Remarks:

cornea 9

Run / experiment:

240 min exposure time

Value:

-0.123

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test material

Irritation parameter:

in vitro irritation score

Remarks:

mean negative control

Run / experiment:

240 min exposure time

Value:

0.442

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: vehicle 0.9% NaCl

Remarks:

stadard deviation ± 0.189

Irritation parameter:

in vitro irritation score

Remarks:

mean positive control

Run / experiment:

240 min exposure time

Value:

89.075

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Positive control 20% Imidazole

Remarks:

standard deviation ± 3.632

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The negative or solvent/vehicle control responses should result in opacity and permeability values, that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. In cases of borderline results in the first testing run, a second testing run should be considered (but not necessarily required), as well as a third one in case of discordant mean IVIS results between the first two testing runs. A result in the first testing run is considered borderline if the predictions from the 3 corneas were non-concordant, such that:
•2 of the 3 corneas gave discordant predictions from the mean of all 3 corneas, OR,
•1 of the 3 corneas gave discordant prediction from the mean of all 3 corneas AND the discordant result was > 10 IVIS units from the cut-off threshold of 55.
•If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean IVIS value), then a final decision can be taken without further testing. If the repeat testing run results in a non-concordant prediction from the initial testing run (based upon the mean IVIS value), then a third and final testing run should be conducted to resolve equivocal predictions, and to classify the test chemical. It may be permissible to waive further testing for classification and labelling in the event any testing run results in a UN GHS Category 1 prediction.
The calculated IVIS value of 0.442 ± 0.189 was well below the cut-off value of 3 (UN GHS no category).
- Acceptance criteria met for positive control: A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean, which was updated at least every three months. . The calculated IVIS value of 89.075 ± 3.632 was within two standard deviations of the current historical mean and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.
- Range of historical values if different from the ones specified in the test guideline:
IVIS: lower and upper limits of acceptance according to OECD Guideline No. 437;
Opacity: upper limit of acceptance according to OECD Guideline No. 437;
Permeability: upper limit of acceptance according to OECD Guideline No. 437.
Historical Control Data (GLP studies of the years 2015 - 2017 (n = 11). Background data are not audited by LPT-QAU.):
-NaCl 0.9%:
*IVIS
Mean: 0.414
Standard deviation: 0.796
Lower limit of acceptance (mean-2*SD ): -1.178
Upper limit of acceptance (mean-2*SD): 2.005
*Opacity
Mean: 0.108
Standard deviation: 0.768
Lower limit of acceptance (mean-2*SD ): -1.428
Upper limit of acceptance (mean-2*SD): 1.643
*Permeability
Mean: 0.024
Standard deviation: 0.012
Lower limit of acceptance (mean-2*SD ): -0.001
Upper limit of acceptance (mean-2*SD): 0.048

-Imidazol 20%
*IVIS
Mean: 84.593
Standard deviation: 12.257
Lower limit of acceptance (mean-2*SD ): 60.079
Upper limit of acceptance (mean-2*SD): 109.108
*Opacity
Mean: 60.730
Standard deviation: 10.816
Lower limit of acceptance (mean-2*SD ): 39.098
Upper limit of acceptance (mean-2*SD): 82.363
*Permeability
Mean: 1.589
Standard deviation: 0.485
Lower limit of acceptance (mean-2*SD ): 0.619
Upper limit of acceptance (mean-2*SD): 2.558

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions IsoQure UR 300 tested in the in vitro BCOP test method, had an IVIS value of 0.285, which is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified for irritation or serious eye damage according to UN GHS classification.

Executive summary:

The purpose of this study was to determine a possible potency of IsoQure UR 300 of being ocular corrosive and severe irritant employing an in vitro system.The Bovine Corneal Opacity and Permeability Assay (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. Three corneas were used for each treatment group (test item, solvent control and positive control). The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% IsoQure UR 300 as recommended in the test guideline 437 for non-surfactant solids. 0.9% NaCl solution was used as the solvent control and 20% Imidazole in 0.9% NaCl solution as the positive control item. The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was 240 minutes. The optical density (OD) was measured at a wavelength of 490 nm. The acceptance criteria of validity were fulfilled in this test. Following treatment with IsoQure UR 300 a mean opacity of 0.305 ± 0.354 and a mean permeability value of <0.001 compared to the negative control were determined. The calculated IVIS of 0.285 ± 0.362 is below the cut-off value of 3 (UN GHS no category). Hence, the test item did not show severely irritant or corrosive properties and consequently it is not classified for irritation or serious eye damage according to UN GHS classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A key in vitro EpiDerm™ study was performed to determine cytotoxic properties of Fenuron to skin cells, which might lead to irritation of human skin, by using an artificial three-dimensional model of human skin (Spruth, 2018a). Fenuron was applied as solid test item to the model skin surface, moistened with Dulbecco’s phosphate buffered saline (D-PBS).D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium. The mean viability of cells exposed to Fenuron was 99.7% of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. Fenuron was considered to be non-cytotoxic and predicted to be non-irritant to skin.

A key Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to determine a possible potency of Fenuron of being ocular corrosive and severe irritant employing an in vitro system. The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% Fenuron as recommended in the test guideline 437 for non-surfactant solids.0.9% NaCl solution was used as the solvent control and 20% Imidazole in0.9% NaCl solution as the positive control item. The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was 240 minutes. The optical density (OD) was measured at a wavelength of 490 nm. Following treatment with Fenuron the calculated IVIS was 0.285±0.362 is below the cut-off value of 3 (UN GHS no category).

Justification for classification or non-classification

Fenuron was non-cytotoxic in an artificial three-dimensional model of human skin, hence, the test item did not show irritant properties and is not classified as for skin corrosion/irritation according to CLP (No. 1272/2008 of 16 December 2008).

Fenuron resulted in an IVIS of 0.285 in the BCOP assay, which is below the cut-off value of 3, hence it is not classified for irritation or serious eye damage according to CLP (No. 1272/2008 of 16 December 2008).