Registration Dossier

Administrative data

Description of key information

Skin

In an in vitro skin irritation assay (recontructed human epidermis) according to OECD 439, the test item was non-irritant to skin.

Eye

In an ex vivo assay using bovine cornea epithelium(BCOP) according to OECD guideline 437, the test item did not induce eye damage, but no final conclusion could be made on the eye irritation potetnial.

In a follow-up in vitro eye irritation test (RhCE) according to OECD guideline 492, a relative cell viability of 104% was determined, indicating no eye irritation potential. Thus, the test item is considered as non-irritant to the eye (UN GHS No Category).

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:
11 December 2017 - 31 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
28 April 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and non-skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:
unchanged (no vehicle)
Details on test system:
EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Supplier: SKINETHIC Laboratories, 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
Batch No.: 17-EKIN-050
Expiry date: 18 December 2017

EpiSkinTMSM KIT Contents

Units: EpiSkinTMSM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EpiSkinTMSM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” for incubations.

The EpiSkinTMSM units were kept in their packaging at room temperature until the pre-incubation was started. The maintenance and assay medium were stored at 2-8°C.

Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material acts directly on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test substance interference with the viability measurement.

As the test item has an intrinsic colour (black), the check-method for possible direct MTT reduction with test item is impossible. The direct interaction with MTT was not defined. However, to avoid the effect of possible interactions with MTT, an additional control was used.

As the test item has an intrinsic colour (black), further evaluation to detect colouring potential was not necessary. Non Specific Colour % (NSC %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
10 mg
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Duration of post-treatment incubation (if applicable):
42 hours (± 1h)
Number of replicates:
In this assay 3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates colour controls and 2 replicates non-specific colour control were used. Furthermore, 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
73
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
68
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
75
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
1-3
Value:
72
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: relative mean tissue viability %
Run / experiment:
1-3
Value:
71
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:
Validity of the Test

The mean OD value of the three negative control tissues was 1.155. The mean OD value obtained for the positive control was 0.075 and this result corresponds to 6 % viability when compared to the results obtained from the negative control. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Possible direct MTT reduction with test item:
As the test item has an intrinsic colour (black), the check-method for possible direct MTT reduction with test item was impossible. The direct interaction with MTT was not defined. However, to avoid the effect of possible interactions with the MTT, an additional control was necessary.The non-specific MTT reduction (NSMTT) was determined to be 0.952 %. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.

Colouring potential of test item:
As the test item has an intrinsic colour (black), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.032. The Non Specific Colour % (NSC %) was calculated as 2.8 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

OD values and viability percentages of the controls:

Substance

Optical Density (OD)

Viability (%)

Negative Control:
1x PBS

1

1.128

98

2

1.264

109

3

1.073

93

mean

1.155

100

standard deviation (SD)

8.49

Positive Control:
SDS (5 % aq.)

1

0.098

9

2

0.064

6

3

0.062

5

mean

0.075

6

standard deviation (SD)

1.77

OD values and viability percentages of the test item:

Test Item

Optical Density (OD)

TODTT

Viability (%)

Relative Viability (%)

Black DB

1

0.841

0.830

73

72

2

0.784

0.773

68

67

3

0.865

0.854

75

74

mean

0.830

0.819

72

71

standard deviation (SD)

3.59

3.59

 

OD values of additional controls for MTT-interacting test item:

Additional controls

Optical Density (OD)

Negative control killed tissues:
1x PBS

1

0.102

2

0.055

3

0.060

mean

0.072

Test item treated killed tissues:
Black DB

1

0.051

2

0.133

3

0.065

mean

0.083

 

OD values and NSC % of additional control:

Additional colour control

Optical Density (OD)

Non Specific Colour %(NSC %)

Black DB
(test item treated tissueswithout MTT incubation)

1

0.040

2.8

2

0.024

mean

0.032

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item has no skin irritation potential.
Executive summary:

An EpiSkinTMSM test with the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1°C for 42 hours in an incubator with 5±1% CO2. The viability of each disk was assessed by incubating the tissues for 3 (±5min) hours with MTT solution at 37±1°C in 5±1% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test item has an intrinsic colour (black), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. The test item is a possible MTT-reducer and has an intrinsic colour (black). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 71 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 October 2017 - 09 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Age at study initiation: at least 9 month old donor cattle
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
The test item was tested as a 20% suspension (w/v) in saline using sonication for 10 minutes. The pH value of the suspension prior to application was 7.69.
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3 corneae per group (test item, negative control, positive control)
Details on study design:
Three corneas were exposed to each 0.75 mL of a 20% (w/v) suspension of the stest item in physiological saline for 240 minutes.
After treatment the test item suspension was rinsed off the corneas and the corneas' opacity was determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.

Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.

For equilibration and prior to application of the test item or controls, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t240).

Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium will be removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values will be determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
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.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
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 – 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 score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)


Criteria for Determination of a Valid Test

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
Run / experiment:
mean
Value:
8.6
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
Replicate 1
Run / experiment:
1
Value:
10.75
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
Replicate 2
Run / experiment:
1
Value:
12.75
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
Replicate 3
Run / experiment:
1
Value:
2.31
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

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

Standard Deviation of in vitro Score

 

 

Mean

 

Mean

 

 

 

 

Negative Control

-2

-1.00

0.082

0.077

-0.77

0.15

Not categorized

1.90

-2

0.059

-1.12

1

0.089

2.34

Positive Control

213.00*

0.006*

213.10

164.26

Category 1

42.44

135.00*

0.090*

136.36

143.00*

0.021*

143.32

Black DB

11.00*

-0.017*

10.75

8.60

No prediction can be made

5.54

13.00*

-0.017*

12.75

2.00*

0.020*

2.31

*corrected values

Interpretation of results:
other: not Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Based on the results of the OECD 437 study, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (UN GHS no Category).
Executive summary:

This in vitro study was performed to assess the corneal damage potential of the test item by means of the BCOP assay using fresh bovine corneas. After a first opacity measurement of the fresh bovine corneas (t0), the 20% (w/v) suspension in saline of the test item Black DB, the positive, and the negative controls were applied to the different corneas and incubated for 240 minutes at 32± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneas and opacity was measured again (t240). After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

For the negative control (saline) an increase of neither opacity nor permeability of the corneas could be observed (mean IVIS = 0.15). The negative control responses resulted 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 control. Therefore the acceptability criteria were fulfilled. The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneas (mean IVIS = 164.26) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The positive control did not met the acceptance criteria of the assay since the mean IVIS is higher than twofold standard deviation of the upper limit of historical established boundaries. However, the raw data show that the IVIS of one cornea out of three of the positive control revealed a higher value compared to the other two corneas which values are in an acceptable range. This single cornea can be declared as outlier. Therefore this deviation has no impact on the validity or the outcome of the study. The Test Item was tested as suspension. Relative to the negative control, the test item caused an increase of the corneal opacity. The calculated mean IVIS was 8.60 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (UN GHS no Category).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2 February- 26 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular™ (OCL-200-EIT) kits are manufactured according to defined quality assurance procedures. All biological components of the EpiOcular™ tissue and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with Triton X-100 (100 μL of 0.3% (v/v) Triton X-100).

Details on environment
The EpiOcular™ human cell construct (MatTek Corporation) is used in this assay. This is a three-dimensional human cornea model.
Supplier: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 821 05 Bratislava, Slovakia
Lot No.: 27022
Expiry date: 08 February 2018

The EpiOcular™ (OCL-200-EIT) units were stored in refrigerator (2-8°C) until the preparation of tissues for treatment is started. The assay media supplied with the kits were stored at refrigerator (2-8°C).

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg


Duration of treatment / exposure:
6 hours (± 15 min) at standard culture conditions (37 °C in an incubator with 5±1 % CO2, ≥95% humidified atmosphere).
Duration of post- treatment incubation (in vitro):
18 hours ± 15 minutes at standard culture conditions.
Number of animals or in vitro replicates:
In this assay 2 replicates per test item and 2 replicates negative controls, 2 replicates positive controls, 2 replicates colour controls and 2 replicates non-specific colour control were used. Furthermore, 2 killed treated tissues and 2 killed negative control tissues are used for the MTT evaluation.
Details on study design:
- Details of the test procedure used :
The tissues were equilibrated to room temperature for about 15 minutes, while the Assay Medium was pre-warmed to 37±1°C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 mL per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37°C in an incubator with 5 ± 1% CO2, ≥95% humidified atmosphere for one hour in the Assay Medium, than the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37°C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 - 24 hours).
During the pre-treatement the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
Approximately 50 mg test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues. The insert was gently shaken from side to side to ensure that tissue was completely covered by the test item. After this procedure the tissue was then returned to its medium.
After rinsing, the tissues were transferred to and immersed in 5 mL RT Assay Medium in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation (Post-Soak) at RT; after it, the medium was decanted off the tissue, and the insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37°C) Assay Medium. The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation).
MTT Test
After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 µL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 10 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, ≥95% humidified atmosphere. Inserts were removed from the 24-well plate after 3 hours ± 10 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol was flowing into the insert. The plate was sealed with parafilm. To extract the MTT, the plates were placed on an orbital plate shaker and shaken (120 rpm) for approximately 2 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative, positive, and colorant controls were treated identically.

- RhCE tissue construct used, including batch number :
EpiOcular™ (OCL-200-EIT), MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 821 05 Bratislava, Slovakia, Lot No 27022
- Doses of test chemical and control substances used
Test item: 50 mg
Control substances: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
Exposure: 6 h
Post-exposure immersion: 25 min
Post-exposure incubation: 18 h
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
The test item has an intrinsic colour (black).
The test item is a possible MTT-reducer.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled):
In this assay 2 replicates per test item and 2 replicates negative controls, 2 replicates positive controls, 2 replicates colour controls and 2 replicates non-specific colour control were used. Furthermore, 2 killed treated tissues and 2 killed negative control tissues are used for the MTT evaluation.
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan :
200 µL sample from each tube was placed into the wells of a 96-well plate and read Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm using isopropanol solutions as the blank (8×200 µL).
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
The irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item. The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60% of the negative control. However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2, further testing with other test methods will be required to decide on its final classification
- Assay Acceptance Criteria:
The mean OD value of the two negative control tissues should be between 0.8 and 2.5.
The acceptable percentage viability for positive control (mean of two tissues) is: 6 hours exposure: below 50 % of control viability for solids
The difference of viability between the two relating tissues of a single chemical is < 20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.
Irritation parameter:
other: mean cell viability %
Run / experiment:
1 and 2
Value:
104
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Table 2: OD values and viability percentages of the controls

Controls

Optical Density (OD)

Viability (%)

Δ%

Negative Control:
Sterile deionized water

1

1.683

96

7.8

2

1.818

104

mean

1.750

100

 

Positive Control:
Methyl acetate

1

0.218

12

2.2

2

0.257

15

mean

0.238

14

 

Table 3: OD values and viability percentages of the test item (including corrected values)

Test Item

Optical Density (OD)

TODTT

Viability (%)

Relative Viability (%)

Δ%

Test Item

1

1.896

1.751

108

100

8.8

2

2.050

1.904

117

109

mean

1.973

1.827

113

104

 

standard deviation (SD)

6.19

6.19

 

  

Table 4: OD values of additional controls for MTT-interacting test item

Additional controls

Optical Density (OD)

Negative control killed tissues:
Sterile deionized water

1

0.046

2

0.050

mean

0.048

Test item treated killed tissues:

1

0.196

2

0.191

mean

0.194

  

Table 5: OD values and NSC % of additional control

Additional colour control

Optical Density (OD)

Non Specific Colour %(NSCliving %)

Δ%

Black DB

1

0.016

1.02

0.2

2

0.020

mean

0.018

 

 

Remark: Δ% is the difference of viability between the two relating tissues

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro eye irritation assay according to OECD guideline 492, a relative mean cell viability of 104 % was determined. The test item is considered as non-irritant to eye (UN GHS No Category).
Executive summary:

In an in vitro eye irritation assay (RhCE) according to OECD guideline 492, the eye irritating potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model was determined. Before treatment the tissues were pre-wetted with ca. 20μL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with 50 mg test item and incubated for 6 hours (± 15 min) at standard culture conditions (37°C in an incubator with 5 % CO2, ≥95% humidified atmosphere). Exposure of test material was terminated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion, test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). The viability of each disk was assessed via MTT assay and quantified spectrophotometrically. Sterile deionized water or methyl acetate treated tissues were used as negative and positive controls, respectively. The test item has an intrinsic colour (bluish black), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. The test item is a possible MTT-reducer and has an intrinsic colour (bluish black). Therefore, a third control for non-specific colour in killed tissues (NSCkilled) was performed with two killed treated tissues to avoid a possible double correction [TODTT (MTT and NSC)] for colour interference.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 104 %). Therefore the test item was considered to be non-irritant to eye (UN GHS No Category).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008 (CLP). As a result the test item is not classified for skin or eye irritation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.