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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 May - 20 July, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model (10 February 2017).
Version / remarks:
10 February 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Phenol, 2,4-dinitro-, sulfurized, thiosulfonated
EC Number:
215-445-8
EC Name:
Phenol, 2,4-dinitro-, sulfurized, thiosulfonated
Cas Number:
1326-83-6
Molecular formula:
not applicable
IUPAC Name:
Reaction product of 2,4-dinitrophenol with polysulfide, thiosulfonated
Test material form:
solid: particulate/powder
Details on test material:
Test item: Solubilised Sulphur Black 1
Appearance: black powder
CAS No: 1326-83-6
Specific details on test material used for the study:
Expiration date: 30 November 2021

Test animals / tissue source

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™ human cell construct (MatTek Corporation) is used in this assay. This three-dimensional human cornea model allows the identification of test items with the potential to induce eye irritation or serious eye damage by assessing cell viability after treatment. The model is composed of stratified human keratinocytes in a three-dimensional structure, consisting of at least three viable layers of cells. Test materials can be applied topically to the model so that also water insoluble materials may be tested. Prior to use, each plate (6, 12, and 24-well) and its cover will be uniquely identified with a permanent marker by a plate number and/or test item number. The cytotoxicity of the test item (and thus the ocular irritation potential) is evaluated by the relative viability of the treated tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, by the succinate dehydrogenase reduction of MTT) in control and test item treated cultures (Berridge, et al., 1996). Data are presented in the form of relative survival (relative MTT conversion).

Quality Control:
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). A certificate of quality as provided by the supplier is annexed to this report.
Number of Replicate Wells.
The condition of the cooler boxes was checked, because the frozen cooler box can guarantee that the temperature of kit was between 2-8°C during the transport.
The cooler boxes were frozen and they were not in direct contact with any of the 24 well plates containing the EpiOcular™ tissues (OCL-200).
The kit was found to be in good order at reception.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg (on top of 0.6 cm² tissue)
Duration of treatment / exposure:
6 hours ± 15 min
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, 90±10% 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.

- RhCE tissue construct used, including lot number
EpiOcular™ (OCL-200-EIT), MatTek In Vitro Life Science Laboratories Mlynské Nivy 73, 821 05 Bratislava, Slovakia; Lot No. 23785

- Doses of test chemical and control substances used
50 mg of the test item or a volume of 50 µL positive control (methyl acetate) or negative control (sterile deionized water) was applied on the tissues surface.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions
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.
The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation).

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
As the test item has an intrinsic colour (dark 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. In addition to the normal procedure, two additional test item-treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer and has an intrinsic colour (dark 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

- Number of tissue replicates used per test chemical and controls
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, 96-well plate spectrophotometer

- Description of the method used to quantify MTT formazan
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 incubato with 5±1 % CO2 protected from light, 90±10% 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 (150 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. Following the formazan extraction, 200 µL sample(s) from each tube (preferably 2×200 µL if possible) was placed into the wells of a 96-well plate (labelled appropriately) 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 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. Depending on the regulatory framework in member countries, the test item is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.

- Complete supporting information for the specific RhCE tissue construct used
see "Any other information on materials and methods"

- Acceptable variability between tissue replicates for positive and negative controls
and acceptable variability between tissue replicates for the test chemical
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:
- 30 minute exposure: below 50% of control viability (liquids)
- 6 hours exposure: below 50% of control viability (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.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: mean tissue viability (%)
Value:
81
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: relative mean tissue viability (%)
Value:
81
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:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes,
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Acceptance Criteria:

The mean OD value of the two negative control tissues was: 1.997

The positive control result showed 7 % viability at 6 hours exposure.

The difference of viability between the two tissue replicates 0.3% to 13.6%

All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Indicator for Potential False Viability

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 0.348 %. As the NSMTT were below 60 % 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 0.012. The Non Specific Colour % (NSC %) was calculated as 0.58 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

Table 3: OD values and viability percentages of the controls

Controls

Optical Density (OD)

Viability (%)

Δ%

Negative Control:
Sterile deionized water

1

1.862

93

13.6

2

2.133

107

mean

1.997

100

 

Positive Control:
Methyl acetate

1

0.070

4

7.6

2

0.222

11

mean

0.146

7

 

Table 4: OD values and viability percentages of the test item (including corrected values)

Test Item

Optical Density (OD)

TODTT

Viability (%)

Relative Viability (%)

Δ%

Solubilised Sulphur Black 1

1

1.745

1.738

87

87

12.1

2

1.504

1.497

75

75

mean

1.624

1.617

81

81

 

standard deviation (SD)

8.53

8.53

 

 

Table 5: OD values of additional controls for MTT-interacting test item

Additional controls

Optical Density (OD)

Negative control killed tissues:
Sterile deionized water

1

0.055

2

0.059

mean

0.057

Test item treated killed tissues:
Solubilised Sulphur Black 1

1

0.056

2

0.071

mean

0.064

 

Table 6: OD values and NSC % of additional control

Additional colour control

Optical Density (OD)

Non Specific Colour %(NSCliving %)

Δ%

Solubilised Sulphur Black 1

1

0.015

0.58

0.3

2

0.009

mean

0.012

 

 

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

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro eye irritation test (RhCE) according to OECD guideline 492, a relative cell viability of 81% was determined, indicating no eye irritation potential under the applied testing conditions. Thus, the test item is considered as non-irritant to eye (UN GHS No Category).
Executive summary:

The eye irritating potential f the test item was determined in an in vitro eye irritation assay (RhCE) according to OECD guideline 492. Two replicates of EpiOcular™ tissues were treated with test item (50 mg) , positive control (methyl acetate, 50 µL) or negative control (sterile deionized water, 50 µL), respectively and incubated for 6 hours (± 15 min) at standard culture conditions (37 °C in an incubator with 5% CO2, 90±10% 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 tissue was assessed by incubating the tissues for 3 hours with MTT solution at 37°C. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

The test item has an intrinsic colour (dark 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. Since the test item is a possible MTT-reducer and has an intrinsic colour (dark black) 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 item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 81 %). 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 eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, the test item is considered as non-irritant to eye (UN GHS No Category).