2,4,6-trimethylbenzene-1,3-diamine

Endpoint summary

Description of key information

The test substance is irritating to skin (LPT, 2016).

Based on the available data no prediction concerning eye irritant or corrosive potential of the test item can be made (LPT, 2016). Subsequently performed eye iritation test based on OECD 492 indicated category 1 classification (serious eye damage) (Fraunhofer, 2021).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-05-02 to 2016-06-24

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 July 28, 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)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be
soluble or insoluble in water.

Vehicle:

other: Dulbecco’s phosphate buffered saline.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:The following Reconstructed Human Epidermis Model was used: EpiDermTM (EPI-200, Lot no. 23338) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- The test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 6.4
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination.
- MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot.
- The ET50 must fall within a range established based on a historical database of results.

NUMBER OF REPLICATE TISSUES: Three replicate tissues were employed

TEST FOR INTERFERENCE OF TEST ITEM WITH MTT REDUCTION ASSAY
- Prior to the testing, the test item was evaluated of colour changes under aqueous conditions. Therefore, 25 mg test item was diluted in
300 µL deionized water and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. At the end of exposure time, the mixture was evaluated of the presence and intensity of the staining. No discolorations were noted.
- Furthermore the test item was evaluated for its potential to interfere with the MTT assay reagent (e.g. reduction). Therefore, 25 mg test item was
dissolved in 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.
Hence, no possible interacting with the MTT measurement had to be considered and no additional test had to be performed.

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



ADMINISTRATION
- 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 moistened with Dulbecco’s phosphate buffered saline to uniformly cover the skin surface.
- The test item is a fine powder. For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.
- Three replicate tissues were employed
- 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)
- The whole exposure period for the used EpiDermTM skin model was 60 minutes
- The incubation conditions were 37°C, 5% CO2 and 95% humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.
- Concurrent negative and positive controls were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue
sensitivity (PC) of the tissues are within a defined historical acceptance range
- Positive control item was 5% aqueous sodium dodecyl sulphate (SDS)
- Negative control was D-PBS5
- 30 μL of negative and positive controls were used

- Viability measurements were not performed immediately after the exposure to the test item, but after a post-treatment incubation period of the
rinsed tissues in fresh medium of 42 hours.
- This period allows both for recovery from weakly irritant effects and for appearance of clear cytotoxic effects
- Each skin sample was placed in an MTT solution of 1 mg/mL (37°C incubation temperature, 5% CO2, 95% humidity) for 3 hours
- The precipitated blue formazan product was extracted using the extraction solution (isopropanol)
- Concentration of the formazan was measured by determining the optical density (OD) at a wavelength of 540 nm in a spectrophotometer
(Tecan Sunrise Magellan Version 6.4)
- Measurements were made for each of the three tissues in 2 replicates

Control samples:

yes, concurrent negative control

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 moistened with Dulbecco’s phosphate buffered saline to uniformly cover the skin surface.
- The test item is a fine powder.
- For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.
- Three replicate tissues were employed.
- 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)

VEHICLE
- Dulbecco's phosphate buffered saline (D-PBS)

NEGATIVE CONTROL
- 30 µL Dulbecco's phosphate buffered saline (D-PBS)

POSITIVE CONTROL
- 30 µL 5% aqueous sodium dodecyl sulphate (SDS)

Duration of treatment / exposure:

An exposure time of 60 minutes was employed.

Duration of post-treatment incubation (if applicable):

Post-treatment incubation period of the rinsed tissues in fresh assay medium of 42 hours.

Number of replicates:

Three replicate tissues were employed.

Species:

other: in vitro

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

60 minutes

Irritation / corrosion parameter:

other: cut-off percentage cell viability

Run / experiment:

The test method is based on reconstructed human epidermis models

Value:

39.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cytotoxic and irritant to skin

Other effects / acceptance of results:

Test for interference of chemicals with MTT
Optical properties of the test item or its chemical action on the MTT may interfere with the assay leading to a false estimate of viability, as the test item may prevent or reverse the colour generation as well as cause it. This may occur when a specific test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.

Prior to the testing, the test item was evaluated of colour changes under aqueous conditions. Therefore, 25 mg test item was mixed with 300 µL deionized water and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. At the end of exposure time, the mixture was evaluated of the presence and intensity of the staining. No discolorations were noted.

Furthermore, the test item was evaluated for its potential to interfere with the MTT assay reagent (e.g. reduction). Therefore, 25 mg test item were mixed with 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.

Hence, no possible interacting with the MTT measurement had to be considered and no additional test had to be performed.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Quality controls (QC) of the model
The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot. The ET50 must fall within a range established based on a historical database of results.


Assay acceptability criteria

Assay acceptance criterion 1: 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 OD570 of the NC tissues is ≥ 1.0 and ≤ 2.5.

Assay acceptance criterion 2: Positive control
A 5% SDS (in H2O) solution was used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than 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%.

Assay acceptance criterion 3: Standard deviation
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%

Interpretation of results

The OD values obtained for each test sample were used to calculate mean percentage viability relative to the negative control, which is set at 100%. The cut-off mean percentage cell viability value that distinguishes irritant from non-classified test substances is given below:

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).

Conclusions:

Under the present test conditions, test item tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was cytotoxic and, hence, irritant to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to determine cytotoxic properties of the test item to skin cells, which might lead to irritation of human skin, by using an artificial three-dimensionalmodel of human skin. TheEpiDerm^TMmodel according to OECD 439 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.

Test item was applied topically as solid test item to the model skin surface, which was moistened with Dulbecco’s phosphate buffered saline. Dulbecco’s phosphate buffered saline (D-PBS) was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item.

 

The mean viability of cells exposed to test item was 39.2% of the negative controls and, hence, was below the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of ≤ 50%. Test item was considered to be cytotoxic and predicted to be irritant to skin in accordance with UN GHS category 2. As test item was found to be non-corrosive in an in vitro skin corrosion test (LPT Report No. 33340) according to OECD guideline 431 distinguishing between Category 1 or Category 2 is admissible.

 

The mean optical density (OD) of the negative control of 3 tissues each was 1.417 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 9.6% 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 required (see section 6.6) were fulfilled.

 

Conclusion

Under the present test conditions, test item tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was cytotoxic and, hence, irritant to skin in an experiment employing an artificial three-dimensional model of human skin.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-03-17 to 2016-04-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Reconstructed Human Epidermis (RHE) Test Method, adopted July 28, 2015.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Vehicle:

water

Remarks:

sterile deionised water

Details on test system:

The following Reconstructed Human Epidermis Model was used:
EpiDerm™ (EPI-200-SCT, Lot no. 23328) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.

Cell viability measurements
- MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1) reduction, which had been shown
to give accurate and reproducible results, was used to measure cell viability
- Each skin sample was placed in an MTT assay solution of 1 mg/mL (37°C incubation temperature, 5% CO2, 95% humidity) for 3 hours
- The precipitated blue formazan product was extracted using the solvent propanol-2, and the concentration of the formazan was measured by
determining the optical density (OD) at a wavelength of 540 nm in a spectrophotometer
- Cell viability measurements were carried out at the end of the exposure period (1st period: 3 min; 2nd period: 60 min). The measurements were
made for each of the two tissues in triplicate.
- Previous checks for interference of the test item with the MTT assay, the nylon mesh or the tissues were performed. No discoloration or test item
interference with the vital dye was noted.

ADMINISTRATION
- EpiDerm tissues were conditioned by pre-incubation (1 hour) in Maintenance Medium1 for release of transport stress related compounds and
debris in the incubator (37°C, 5% CO2, 95% humidity).
- After pre-incubation tissues were transferred to fresh Maintenance Medium and topically exposed with the test chemicals for 3 min and 1 h,
respectively.
- Two tissues were used per treatment, negative and positive control and exposition time (12 tissues in total).
- 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface which was moistened with
sterile deionised water to ensure adequate contact with the skin. A minimum of 30 mg or 70 μL substance applied per cm2 is required by the
guidelines.
- Positive control item was 8 N KOH4, 50 µl
- Negative control was sterile deionised water, 50µl
- After exposure tissues were rinsed, blotted and assay medium was replaced by MTT assay medium2 (final concentration: 1 mg
MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue)/mL medium).
- After 3-h incubation, tissues were washed with Dulbecco's phosphate buffered saline (D-PBS), blotted and the blue formazan salt was extracted with propanol-2.
- The optical density of the formazan extract was determined spectrophotometrically at 540 nm and cell viability was calculated for each tissue as
% of the mean of the negative control tissues.
- Skin corrosion potential of the test materials was classified according to the remaining cell viability obtained after 3 minutes or 1 hour exposure
with the test chemical.

Control samples:

yes, concurrent vehicle

Amount/concentration applied:

- Test item: 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface which was
moistened with sterile deionised water to ensure adequate contact with the skin.
- Positive control item was 8 N KOH4, 50 µl
- Negative control was sterile deionised water, 50µl
- 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).

Duration of treatment / exposure:

3 minutes and 1 hour

Species:

other: in vitro

Irritation / corrosion parameter:

other: cut-off percentage cell viability value

Run / experiment:

The EpiDerm™ model was employed, 3 minute exposure

Value:

114.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Under the present test conditions test item tested at two exposure periods of 3 minutes (cell viability value =114.8 %) or 1 hour (cell viability value =19.2 %) was non-corrosive to skin.

Irritation / corrosion parameter:

other: cut-off percentage cell viability value

Run / experiment:

The EpiDerm™ model was employed, 1 hour exposure

Value:

19.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Under the present test conditions test item tested at two exposure periods of 3 minutes (cell viability value =114.8 %) or 1 hour (cell viability value =19.2 %) was non-corrosive to skin.

Assay acceptability criteria

Assay acceptance criterion 1: Negative control

The absolute OD of the negative control (NC) tissues (treated with sterile deionised water) 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 tissues treated with the negative control should not be below historically established boundaries.

The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 0.8 and ≤ 2.8.

Assay acceptance criterion 2: Positive control

A8N KOHwas used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay.

Tissues treated with the PC, should reflect the ability of the tissues to respond to a corrosive chemical under the conditions of the test method (viability after 1hour exposure: < 15%).

Assay acceptance criterion 3:variability between tissue replicates

Associated and appropriate measures of variability between tissue replicatesshould not exceed 30% (in the range of 20 – 100% viability).

Interpretation of results

The OD values obtained for each test sample was used to calculate a percentage viability relative to the negative control, which is arbitrarily set at 100%. The cut-off percentage cell viability value distinguishing corrosive from non-corrosive test items (or discriminating between different corrosive classes, e.g. subcategories 1A, 1B and 1C, or the statistical procedure(s) used to evaluate the results and identify corrosive materials, is clearly defined and documented, and shown to be appropriate. The criteria of corrosivity associated with the EpiDermTM model are as follows:

- the test item is considered to be corrosive to skin and classified as category 1 (or optional category 1A8), if the viability after 3

minutes exposure is less than 50%;

- the test item is considered to be corrosive to skin and classified as sub-category 1B-and-1C, if the viability after 3 minutes

exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%;

- the test item is considered to be non-corrosive to skin, if the viability after 3 minutes exposure is greater than or equal to 50% and

the viability after 1 hour exposure is greater than or equal to 15%.

Conclusions:

Under the present test conditions test item tested at two exposure periods of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to assess the corrosive properties of test item to human skin,in anexperiment with an artificial three-dimensional model of human skin. TheEpiDerm™model according to OECD 431 was employed.

 

Two tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined byusing 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.

Test item was applied topically as solid test item to the model skin surface, which was moistened with sterile deionised water. Sterile deionised water was used as the negative control. 8N KOH was used as the positive reference item. The test item and the reference items were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 114.8% after a 3-minute exposure period and 19.2% after an 1‑hour exposure.The 3-minute and the1-hour exposure values were above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of ≥ 50% and ≥ 15%, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean optical density (OD) of the negative control of 2 tissues was 1.960 (3‑minute exposure) or 2.243 (1-hour exposure) and was well within the acceptable range of ≥ 0.8 to ≤ 2.8. The viability of cells treated with the positive reference item 8N KOH were 8.4% (3-minute exposure) and 7.1% (1-hour exposure) of the negative control and, hence well below the 15% cut-off value at the 1-hour exposure. The difference of viability between the two tissue replicates(at 20 - 100% viability) was below the limit of acceptance of 30%. Hence, all acceptance criteria were fulfilled.

 

Conclusion

Under the present test conditions test item tested at two exposure periods of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2021; finalised 12.01.2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

According to the guidance document No 263 on INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) for serious eye damage and eye irritation non OECD adopted in vitro test methods can be conducted for an weight of evidence analysis. If no decision regarding classification and labelling can be drawn according to the performed guideline tests, other in vitro test methodes for serious eye damage & eye irritation can be conducted even. As mentioned in the IATA 263 these in vitro test methods not even need to be adopted by the OECD.

Qualifier:

equivalent or similar 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:

adopted June 14, 2019

Deviations:

yes

Remarks:

used cornea model: inhouse developed reconstructed cornea model ( RCE)

GLP compliance:

no

Specific details on test material used for the study:

solid

Species:

human

Details on test animals or tissues and environmental conditions:

The in vitro eye irritation test ( is based on the protocol of the OECD test guideline 492 with the Fraunhofer-in house developed reconstructed cornea model (RCE).

All experiments were conducted in compliance with the rules for investigation on human subjects, as defined in the Declaration of Helsinki. Informed consent was obtained from all subjects prior to the study. Human corneal cells were isolated from spare limbal rings and from corneas that failed the quality criteria for clinical use in accordance and with the approval of the local ethics committee (Ethik-Kommission der Universität Würzburg, approval number 182/10) and the informed consent of the patients for the study participation. Corneal biopsies were provided by the eye clinic of the Universitätsklinikum Würzburg (Würzburg, Germany). No tissues were procured from prisoners.
Corneal tissues were transferred and washed in a petri dish with phosphate-buffered saline. Subsequently, the cornea was cut into horizontal stripes of about 2–3 mm and put in a petri dish with dispase [2 U/ml] (Thermo Fisher Scientific, Waltham, MA USA)) for 18 hours at 4 °C. The epithelium was stripped off the central cornea to the limbus with forceps and collected in a new petri dish with fresh phosphate-buffered saline. The epithelial sheets were centrifuged and reduced to small pieces by pipetting for cell seeding. The corneal epithelium was cultured in corneal epithelial cell medium (LGC Standards, Wesel, Germany). The RCE was generated from human corneal epithelial cells. The cells were seeded as described above with a cell density of 2.5 × 105 cells/cm2 and cultured for 11 days.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg solid
50 µl liquid (positive and negative control)

Duration of treatment / exposure:

liquids: 30 ± 2 min
solids: 6 + 0.25 h

Observation period (in vivo):

up to 11 days

Duration of post- treatment incubation (in vitro):

liquids: 120 ± 15 min
solids: 18 + 0.25 h

Number of animals or in vitro replicates:

2x RCE were used for each assays and treatment group (test item, positive and negative control), 12x in vitro corneas in total.

Details on study design:

The in vitro eye irritation test ( is based on the protocol of the OECD test guideline 492 with the in house developed reconstructed cornea model (RCE). The EIT is complemented by the integration of impedance spectroscopy for long term observation of the applied tissue models. The RCE are pre-wet with 20 μl of phosphate buffered saline for 30 min. Each assay and each treatment (test item, positive & neg. control) were conducted in duplicate. Therefore,12 RCE were used in total. Duplicates of cornea epithelia models, are exposed for 30 min ± 2 min (liquids) or 6 h ±15 min (solids) via topical application of the test material with 50 μl (liquids) or 50 mg (solids) at 37 C, 5% CO2 and 95% humidity in an incubator. Tissue models treated with water (CAS RN 7732 18 5) are used as negative control or 10% Benzalkonium chloride (CAS RN 63449 41 2 as positive control. After exposure the test substances are rinsed through three washing steps at room temperature (RT). An additional post-soak of 12± 2 min (liquids) or 25± 2 min (solids) ensures the removal of excess substance by immersing the models in 5 ml Epilife Medium (Life Technologies, MEPI 500 CA) at RT. Subsequently, viability is measured after post incubation of 120 ± 15 min (liquids) or 18 ±0.25 h (solids) at 37C, 5% CO2 and 95% humidity. Transepithelial electrical resistance at 1000 Hz (TEER 1000 Hz) measurements are performed with a customized device. Barrier integrity of tissue models are evaluated via impedance spectroscopy prior to substance application and after the post incubation and is repeated on day 1, 3, 7 and 11 after test material application. In this study the test protocol for solids was applied. Acceptability range for negative control before treatment 4000 >NC <600 Ohm/cm².

The following destructive endpoint measurements are performed:
- Tissue viability of two RCE is quantified by MTT reduction after post incubtaion
The following non destructive measurements are performed for each test material:
- Barrier integrity of two RCE is quantified by impedance spectroscopy before application and after application at day 0 1 3 7 and 11

VIABILITY measurments (MTT):
The assessment of model viability was conducted based on the protocol as described in OECD test guideline 492. Briefly, reconstructed corneal epithelial models were placed in 200 μl MTT solution 1 mg/mL in PBS)(Sigma Aldrich, M 2128 1 G) for 3 h in a humidified atmosphere with 37C and 5% CO2. The MTT reduction was then quantified by extracting the precipitated blue formazan salt with 2 ml 2-propanol and measuring the optical density of the extract at a wavelength at 570 nm using a spectrophotometer (InFinite M Nano, Tecan). After correcting thedata using 2-propanol as blank the relative tissue viability was calculated for each tissue by normalizing the corrected optical density values to the negative control which was set to 100. Acceptability range for negative control 2.5> OD> 0 8.

IMPEDACE SPECTROSCOPY (TEER 1000 Hz):
Impedance spectroscopy allows for a noninvasive evaluation of a tissue model of interest by regarding the tissue’s electrical characteristics Employing a measuring system developed by the Fraunhofer one can identify biologically relevant electrical parameters from impedance spectra in a frequency range from 1 Hz to 100 kHz. Experimental studies revealed a strong correlation between these electrical parameters and the state of the tissue models (Groeber et al ., 2015) . Compared to conventional methods, impedance spectroscopy shows a higher sensitivity in the detection of perturbations within the non viable cell layers. Therefore, an electrode is installed on top and a second one underneath a model and a low current is introduced. Since the walls of the carrier inserts are nonconductive, the complex alternating current resistance (impedance) can be yielded from the sloping voltage at a certain frequency point It could be asserted that the impedance at 1000 Hz multiplied by the culture area of the carrier inserts suits best answering the initial issues of the present study. This transepithelial electrical resistance (TEER) at 1000 Hz (TEER 1000 Hz) can be measured repeatedly during culture, which allows that the exact same models can be compared as this measuring technique works noninvasive. The results are presented in relative values normalized to the negative control. Barrier integrity of tissue models are evaluated via impedance spectroscopy prior to substance application and after the post incubation and is repeated on day 1, 3, 7 and 11 after test material application.

Irritation parameter:

TEER value (Transepithelial Electrical Resistance) 

Remarks:

at 1000 Hz

Run / experiment:

impedance spectroscopy at 1000 Hz day 7d

Value:

14

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

< 50% indicating a Category 1 substance by TEER measurement

Irritation parameter:

TEER value (Transepithelial Electrical Resistance) 

Remarks:

at 1000 Hz

Run / experiment:

impedance spectroscopy at 1000 Hz at day 1

Value:

50

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

50% < 60% cut-off

Irritation parameter:

mean percent tissue viability 

Remarks:

% of negative control

Run / experiment:

MTT

Value:

1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

category 1 or 2 by viability

Other effects / acceptance of results:

ACCEPTANCE of RESULTS:
- Acceptance criteria met for viability assay: yes
- Acceptance criteria met for TEER measurments: yes
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

	Individual values of in vitro Eye Irritation Viability
Treatment	Replicate	individual raw OD540 nm	individual OD540 nm blank corrected	mean OD540 nm (SD)	OD540 nm of 2 tissues (SD)	individual Viability % (SD)	mean Viability % (SD)
Negative control	tissue 1	1.2433	1.2017	1.2473	1.2793	97.50	100.00
Water		1.3344	1.2928	(0.06)	(0.05)		(3.54)
	tissue 2	1.3226	1.281	1.3113		102.50
		1.3831	1.3415	(0.04)
Positive control	tissue 1	0.0456	0.004	0.0042	0.3244	0.32	0.36
10 % benzalkonium chloride		0.0459	0.0043	(0.0002)	(0.0006)		(0.05)
	tissue 2	0.0464	0.0048	0.0050		0.39
		0.0468	0.0052	(0.0003)
Test item	tissue 1	0.0549	0.0133	0.0122	0,00885	0.95	0.69
		0.0527	0.0111	(0.002)	(0.0047)		(0.37)
	tissue 2	0.0475	0.0059	0.0055		0.43
		0.0467	0.0051	(0.0006)
Blank	tissue 1	0.0408		0.0416
		0.0424
OD:	Optical density
SD:	Standard deviation

 

 

Table 2:

Individual values of in vitro Eye Irritation Transepithelia electrical resistance at 1000 Hz
		individual raw TEER1000Hz data black corrected			individual normalized TEER %
Treatment	Timepoint			TEER1000Hz of 2 tissues			TEER % of 2 tissues
		tissue 1	tissue 2		tissue 1	tissue 2	mean	SD
Negative control	Day 0 before	970.0	1126.5	1048.2	92.54	107.46	100.00	10.6
Water	Day 0 after	944.2	1078.3	1011.2	93.37	106.63	100.00	9.4
	Day 1	920.7	934.5	927.6	99.26	100.74	100.00	1.1
	Day 3	990.4	1179.9	1085.1	91.27	108.73	100.00	12.3
	Day 7	1719.1	1639.3	1679.2	102.38	97.62	100.00	3.4
	Day 11	2813.8	N/A	2813.8	100.00	N/A	100.00	N/A
Positive control	Day 0 before	1611.5	1625.4	1618.4	153.73	155.06	154.40	0.9
10 % benzalkonium chloride	Day 0 after	22.3	7.8	15.1	2.21	0.77	1.50	1
	Day 1	15.9	11.1	13.5	1.71	1.20	1.50	0.4
	Day 3	-11.9	-1.1	-6.5	1.10	-0.10	-0.60	0.7
	Day 7	3.6	4.6	4.1	0.21	0.28	0.20	0
	Day 11	43.8	N/A	43.8	1.56	N/A	1.60	N/A
Test Item	Day 0 before	1182.3	1087.9	1135.1	112.76	103.78	108.30	6.4
	Day 0 after	384.1	623.9	504	37.98	61.70	49.80	16.8
	Day 1	301.9	525.2	413.6	32.55	56.62	44.60	17
	Day 3	265.7	338.8	302.2	24.28	31.22	27.90	4.8
	Day 7	286.8	165.6	226.2	17.08	9.86	13.50	5.1
	Day 11	186.2	N/A	186.2	6.62	N/A	6.60	N/A
SD:	Standard deviation

 

Conclusions:

The test substance was evaluated in an in vitro eye irritation test based on the OECD testing guideline 492 with the Fraunhofer-inhouse developed reconstructed cornea model ( RCE).

Viability was assessed by an MTT assay and barrier function by transepithelial electrical
resistance at 1000Hz (TEER 1000Hz). The viability test shows an irritative effect of the testing substance, suggesting a category 1 or 2 substance.
The TEER 1000Hz indicates a persistent irritative effect over time, indicating a category 1
substance (serious eye damage).

Executive summary:

The test item was evaluated in an in vitro eye irritation test based on the OECD test guideline 492 with the Fraunhofer inhouse developed reconstructed cornea model (RCE).
• Viability was assessed by an MTT assay and barrier function by transepithelial electrical resistance at 1000Hz (TEER 1000Hz).
• the test item was tested as solid substance and was incubated for 6 hours after topical application. Tissue models treated with water (CAS RN: 7732-18-5) were used as negative control or 10 % Benzalkonium chloride (CAS RN: 63449-41-2) as positive control Eight models were used in total
• Internal Acceptance criteria of NC for MTT (2.5>NC>0.8) and TEER (4000>NC>600 ) measurement were met
• the test item revealed a reduction of viability below 60%. As the measured viability was 1% after application of the test item, indicating a category 1 or 2 substance for eye irritation by viability.
• the test item demonstrated a decrease in TEER 1000Hz below 60% with 50% after application that persisted at day 7 with a TEER 1000Hz below 50 % with 14%, indicating a category 1 substance by TEER measurement.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-03-17 to 2016-04-20

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 Ocular Corrosives and Severe Irritants)

Version / remarks:

adpted July 26, 2013

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:

December 09, 2010

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% (w/v).

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. To minimize deterioration and bacterial
contamination, 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.
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.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

750 µl test item, 20% suspension in 0.9% sodium chloride solution (w/v
750 µl solvent control item, 0.9% sodium chloride solution
750 µl positive control item, 20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution

Duration of treatment / exposure:

Exposure period: 240 minutes

Duration of post- treatment incubation (in vitro):

After rinsing the corneas were incubated at 32°C ± 1°C for 90 ± 5 minutes. After this post incubation period, the corneas were examined.

Number of animals or in vitro replicates:

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

Details on study design:

PREPARATION OF BOVINE EYES
- Corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium)
chambers
- The chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM)
- Corneal holder was equilibrated at 32 ± 1°C for at least one hour
- After equilibration period, fresh pre-warmed EMEM was added to both chambers
- 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
- 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 solvent control corneas
- The remaining corneas were then distributed into treatment, solvent and positive control groups
ADMINISTRATION
- Three corneas were used for each treatment group
Negative control item: 0.9% sodium chloride solution
Positive control item: 20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution
Test item: 20% suspension in 0.9% sodium chloride solution (w/v)
- Exposure period: 240 minutes
- After exposure period of 240 minutes the test item, solvent control and positive control, were removed from each chamber and epithelium was
washed with EMEM at least three times
- Subsequently, 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
- Corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber
- Chamber was then filled with EMEM without phenol red
EXAMINATION
- Corneal injury was assessed by evaluating the opacity and permeability of the cornea
- 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
- 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
- Amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader
(Tecan Sunrise Magellan Version 6.412).
- 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 using a standard 1 cm path length

Irritation parameter:

in vitro irritation score

Run / experiment:

BOVINE CORNEAL OPACITY AND PERMEABILITY TEST

Value:

36.07

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: no prediction concerning irritant or corrosive potential of the test item can be made.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Opacity Values

	Cornea No.	Opacity [Opacity Units]	Corrected Opacity
				Mean of group	Standard deviation
0.9% NaCl	1	-0.398	-0.544	-0.544	0.128
	2	-0.597
	3	-0.637
20% Imidazol	4	68.113	44.010	75.205	10.188
	5	70.623	47.476
	6	86.879	57.397
20% Test item	7	3.651	5.404	2.855	0.708
	8	2.297	2.297
	9	2.616	2.616

Permeability OD Values (490 nm)

	Cornea no.	Permeability [OD]	Mean of Triplicates	Corrected Permeability [OD]
					Per Cornea		Per Group
					Mean	SD	Mean	SD
0.9% NaCl	1	-0.001	0.001	-			0.027	0.031
		0.000		-
		0.003		-	0.001	0.002
	2	0.063	0.061	-	0.061	0.002
		0.061		-
		0.060		-
	3	0.017	0.019	-	0.019	0.002
		0.018		-
		0.021		-
20% Imidazol	4	1.132	1.143	1.105	1.116	0.015	0.948	0.200
		1.138		1.111
		1.160		1.133
	5	0.767	0.754	0.740	0.727	0.012
		0.743		0.716
		0.753		0.726
	6	1.006	1.028	0.979	1.001	0.023
		1.028		1.001
		1.051		1.024
20% Test item	7	2.175	2.185	2.148	2.158	0.008	2.214	0.049
		2.190		2.163
		2.189		2.162
	8	2.282	2.276	2.255	2.249	0.006
		2.270		2.243
		2.275		2.248
	9	2.264	2.263	2.237	2.236	0.021
		2.283		2.256
		2.242		2.215

SD : standard deviation

OD : optical density

In vitro irritancy score (IVIS)

	Cornea No.	Opacity	Permeability	IVIS
				Per Cornea	Per Group
					Mean	SD
0.9% NaCl	1	-0.398	0.001	-0.383	-0.139	0.396
	2	-0.597	0.061	0.318
	3	-0.637	0.019	-0.352
20% Imidazol	4	68.113	1.116	84.853	89.425	10.926
	5	70.623	0.727	81.528
	6	86.879	1.001	101.894
20% Test item	7	3.651	2.158	36.021	36.070	0.075
	8	2.297	2.249	36.032
	9	2.616	2.236	36.156

Conclusions:

Under the present test conditions, test item tested in the in vitro BCOP test method, had an IVIS value of 36.070, which is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently no prediction concerning irritant or corrosive potential of the test item can be made.

Executive summary:

The purpose of this study was to determinea possible potency of test item of being 'ocular corrosive and severe irritant' employing an in vitro system.The Bovine Corneal Opacity and Permeability Assay (BCOP) test method according to OECD 437 is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, possible damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability inisolated corneas from bovine eyes.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber.The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

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% test item 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 test item a mean opacity value of 2.855 ± 0.708 and a mean permeability value of 2.214 ± 0.049 compared to the solvent control were determined. An IVIS of 36.070 ± 0.075 was calculated. The calculated IVIS of 36.070 is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently no prediction concerning irritant or corrosive potential of the test item can be made.

 

Conclusion

Under the present test conditions, test item tested in the in vitro BCOP test method, had an IVIS value of 36.070, which is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently no prediction concerning irritant or corrosive potential of the test item can be made.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Under the present test conditions the test item tested according to OECD 431 at two exposure periods of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin (LPT, 2016). Additionally, under the present test conditions, the test item tested according to OECD 439 at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was cytotoxic and, hence, irritant to skin in accordance with UN GHS category 2 in an experiment employing an artificial three-dimensional model of human skin (LPT, 2016).

Under the present test conditions, test item tested in the in vitro BCOP test method, had an IVIS value of 36.070, which is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1). Based on these gudieline tests, no prediction concerning irritant or corrosive potential of the test item can be made.

Subsequently, the test substance was tested in an eye irritation test based on OECD 492 with the Fraunhofer-In-house developed reconstructed cornea model (RCE). Viability was assessed by an MTT assay and barrier function by transepithelial electrical resistance at 1000Hz (TEER 1000Hz). The viability test shows an irritative effect of the testing substance, suggesting a category 1 or 2 substance. The TEER 1000Hz indicates a persistent irritative effect over time, indicating a category 1 substance (serious eye damage) (Fraunhofer, 2021).

 

Justification for classification or non-classification

Based on the assessment of two in vitro studies (skin corrosion and skin irritation) and according to criteria of EC Regulation 1272/2008 the test item is irritating to skin and therefore must be classified as Skin Irrit. Cat. 2; H315: "Causes skin irritation.".

Based on an available guideline test according to OECD 437, no unequivocal prediction concerning eye irritant or corrosive potential of the test item can be made. Based on an in vitro eye irritation test based on the OECD test guideline 492 with the inhouse developed reconstructed cornea model (RCE), the test compound must be classified as Category 1 substance causing seroius Eye damage according to criteria of EC Regulation 1272/2008.