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Description of key information

Skin irritation: not irritating (OECD 439, GLP; OECD 404, GLP)

Eye irritation: not irritating (OECD 437, GLP; OECD 405, GLP)

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:
2017-07-04 to 2017-08-18
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:
2015-07-28
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-09-14
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: humans
Source strain:
not specified
Details on animal used as source of test system:
not applicable
Justification for test system used:
In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
other: Dulbecco's phosphate buffered saline
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin model (source: MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue lot number: 25837
- Delivery date: 2017-08-15

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37 ± 1.5 °C
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 minutes, room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were rinsed with DPBS at least 15 times in order to remove any residual test material.

After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were again rinsed with DPBS. The tissues were then transferred into plates with assay medium. Tissues were incubated for 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation medium was changed (pre-warmed fresh medium). Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was 41 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/ well)
- Incubation time with MTT: 3 hours
- Extraction of Formazan: after the incubation period, the tissues were rinsed three times with DPBS. The tissues were transferred into new plates containing extractant solution (isopropanol) in each well ensuring that the tissues were completely covered and the plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for 2 hours and 40 minutes while shaking at room temperature.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken and the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. The optical density was read with a microplate reader. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm

TEST FOR COLOUR INTERFERENCE
Before the test started, a functional check for colour interference was performed. 25 ± 2 mg of the test item were added to deionised water. The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 minutes. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated.

TEST FOR DIRECT MTT REDUCTION
The test item was evaluated for its potential to interfere with the MTT assay. To test if a test item directly reduces MTT, 25 ± 2 mg of the test item were added to 1 mL of the MTT-solution (1mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1- virus, Hepatitis B virus, and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: relative viability(%) = (mean OD test item or positive control/ mean OD of negative control) x 100
For the test item and the positive control, the mean relative viability ± relative standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model: if the mean relative tissue viability of three individual tissues is less or equal to 50% of the negative control, the test item needs to be classified and labeled for its skin irritation potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of the test item, wetted with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% Sodium lauryl Sulfate (SLS) solution
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
41 hours
Number of replicates:
triplicates
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Value:
95.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: the test item did not dye water or did not change colour in the presence of water. An additional test with viable tissues (but without MTT addition) was not necessary to be performed in the main experiment.
- Direct-MTT reduction: the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed in the main experiment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control, the absorbance values (1.317, 1.368, and 1.325 (mean: 1.337)) were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 4.4 % (acceptability criterion: positive control is ≤ 20 %).
- Acceptance criteria met for variability between replicate measurements: the relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 7.7 % (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: ≤ 18%).
Please refer to the field "Any other information on results incl. tables" below

Table 1: Results after treatment with N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide and the controls

Dose Group

Tissue No.

Absor-bance 570 nm
Well 1

Absor-bance 570 nm
Well 2

Absor-bance 570 nm
Well 3

Mean Absor-bance of 3 Wells

Mean-Absor-bance

of three wells blank

corrected

Mean

Absor-bance

of 3 tissues

after blank correction

Rel. Viability [%] Tissue 1, 2, 3*

Relative Standard Deviation

[%]

Mean Rel. Viability

[%]**

Blank

 

0.038

0.038

0.038

0.038

 

Negative Control

1

1.349

1.359

1.357

1.355

1.317

1.337

98.6

2.0

100.0

2

1.415

1.405

1.396

1.405

1.368

102.3

3

1.374

1.356

1.358

1.363

1.325

99.1

Positive Control

1

0.087

0.092

0.094

0.091

0.053

0.058

4.0

7.7

4.4

2

0.098

0.098

0.098

0.098

0.060

4.5

3

0.100

0.099

0.099

0.100

0.062

4.6

Test Item

1

1.270

1.389

1.378

1.346

1.308

1.275

97.9

2.3

95.4

2

1.301

1.287

1.298

1.295

1.258

94.1

3

1.297

1.309

1.283

1.296

1.258

94.2

* relative viability [rounded values]: 100 x (absorbance test item/positive control/negative control)/ mean absorbance negative control

** mean relative viabiliy [rounded values]:100 x (mean absorbance (test item/ positive control/negative control))/ (mean absorbance (negative control))

Table 2: Historical data

Positive Control

Negative Control [OD570]

Mean Viability

4.37%

Mean Absorption

1.74

Rel. Standard Deviation

21.60%

Rel. Standard Deviation

9.40%

Range of Viabilities

2.20% - 6.78%

Range of Absorbance

1.34– 2.00

Mean Absorption

0.08

 

Rel. Standard Deviation

20.12%

Range of Absorbance

0.03 - 0.11

Data of 103 studies performed from July 2015 until March 2017.

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide is not irritant to skin according to UN GHS and EU CLP (Regulation (EC) 1272/2008 and subsequent regulations) regulation.
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:
2017-09-19
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:
2013-07-26
Deviations:
no
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:
2010-12-09
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details
Version / remarks:
April 1997
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-09-14
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co.KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: corneae were isolated and used on the same day after delivery of the eyes
Vehicle:
other: 0.9% (w/v) NaCl in deionised water
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration: 20 % suspension (w/v) in vehicle

The test item was test as suspension in the vehicle using sonication for 10 minutes.
Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
not required
Number of animals or in vitro replicates:
Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
Details on study design:
PREPARATION OF CORNEAS
- each isolated cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with incubation medium cMEM (MEM, supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin and 1 % fetal calf serum).
- for equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS
- all eyes were carefully examined macroscopically for defects before removing the cornea. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- at the end of the equilibration period of the corneae in the holder, the basal opacity was determined (t0).
- each corneae with a value of the basal opacity > 7 was discarded.

APPLICATION DOSE AND EXPOSURE TIME
- the anterior compartment received the test item suspension or negative or positive control at a volume of 0.75 mL each on the surface of the corneae.
- corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath (incubation time: 240 minutes).
- after the incubation time, the test item or control items were rinsed off with saline.
- fresh incubation medium was added into the anterior compartment and opacity was measured (t240).
- permeability of the corneae was determined.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the opacitometer (OP_KiT opacitometer (Electro Design)) was calibrated and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Evaluation of opacity:
- the change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
- the average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (Versamax® Molecular Devices)(OD490).
- after the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by 0.5% (w/v) sodium fluorescein solution in HBSS.
- corneae were incubated in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C.
- incubation medium from the posterior compartment was removed, mixed and the optical density at 490 nm was determined with a microplate reader.
Evaluation permeability:
- the corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the IVIS score obtained, the test item is classified into the following category according to OECD guideline 437 (please refer to table 1 in the field "Any other information on material and methods incl. tables" below).

DECISION CRITERIA:
The test will be acceptable if:
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
Irritation parameter:
in vitro irritation score
Value:
0.28
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- for the negative control (saline) an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.31).
- the positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =122.90) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Please refer to the field "Any other information on results incl. tables" below.

Table 1: Results after 240 Minutes Treatment Time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposed in vitro Irritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

0

0.33

0.074

0.065

1.11

1.31

No Category

1

0.061

1.92

0

0.061

0.92

Positive Control

118.67*

-0.011*

118.50

122.90

Category 1

122.67*

0.005*

122.74

127.67*

-0.014*

127.45

Test item

-0.33*

0.021*

-0.02

0.28

No Category

-0.33*

0.019*

-0.05

0.67*

0.016*

0.90

*corrected values

Table 2: Historical Data

 

Positive Control

Negative Control

Mean IVIS

112.88

1.32

Standard Deviation of IVIS

9.15

0.19

Range of IVIS

98.30—127.22

1.01—1.64

95 % Control limits of IVISpos

94.58—131.17

 

Mean Opacity t240min

111.11

0.20

Standard Deviation of
Opacity t240min

12.20

0.19

Range of Opacity t240min

82.00—131.67

0.00—0.33

Mean Permeability

0.12

0.07

Standard Deviation of Permeability

0.13

0.01

Range of Permeability

-0.01—0.52

0.07—0.09

Values of 31 studies with solid test items sharing 15 sets of controls, performed between January 2017 and July 2017.

Interpretation of results:
GHS criteria not met
Conclusions:
Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 0.28. According to OECD 437 and in accordance with Regulation (EC) 1272/2008 and subsequent regulations the test item is not categorized (GHS).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin irritation:

The substance was not observed to be irritating to the skin in a reliable in vitro skin irritation study according to OECD 439.

Eye irritation:

The substance was not observed to be irritating to the eye in a reliable in vitro eye irritation study according to OECD 437.

Justification for classification or non-classification

Skin irritation:

The substance does not possess a skin irritation potential according to an in-vivo OECD 404 and an in-vitro OECD 439 test and does not require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

The substance does not possess an eye irritation potential according to an in-vivo OECD 405 and an in-vitro OECD 437 test and does not require classification as eye irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

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