Reaction products of stearic acid with 2-aminoethanol, maleic anhydride and sodium sulphite

Endpoint summary

Description of key information

- Not irritating to skin (GLPs and OECD 431 and OECD 439 compliant studies)

- No irritating to eyes (GLPs and OECD 437 compliant study)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-04 - 2018-02-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified (adult donors)

Justification for test system used:

The corrosivity potential of a chemical may be predicted by measurement of its cytotoxic effect, as reflected in the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] assay, on EPISKINTM(SM) reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD No. 404) and is specifically approved as a replacement for the in vivo skin corrosivity test within OECD No. 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM(SM)
- Tissue batch number(s): 17-EKIN-050
- Production date: 12 December 2017
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 14 December 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (22.4-25.1°C)
- Temperature of post-treatment incubation: N/A

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: one washing with PBS solution
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT
- Incubation time: 3 hours (±15 minutes)
- Spectrophotometer: Thermo Fisher Scientific
- Wavelength: 570 nm.
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 24072800, Serial
Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Quality control: The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkinTM(SM) Test Kits used in the present study) .
- Contamination: All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.


NUMBER OF REPLICATE TISSUES: Two replicates for test item were used. Two negative controls and two positive controls were also run in this assay.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : duplicate
- Method of calculation used: Non specific MTT reduction calculation (NSMTT%):
NSMTT% = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD
ODKT: test item treated killed tissues OD
ODNC: negative control OD


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

PREDICTION MODEL / DECISION CRITERIA:
The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).
For 2 disks:
If both disks have mean viability of ≥35% = Non Corrosive
If both disks have mean viability of <35% = Corrosive (at the corresponding incubation period)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The test item was applied as supplied, no formulation was required.
The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- ~95 mg of test item was applied evenly to the epidermal surface of each of two test units and each additional control skin units and then 100 μL physiological saline was added to the test item to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (22.4-25.1°C) covered with the plate lids.

Duration of post-treatment incubation (if applicable):

Rinsing:
After the incubation time (4 hours), all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly.
The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MMT test:
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two living colour control units). The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 in a >95% humidified atmosphere for 3 hours (±15 minutes), protected from light.

Formazan extraction:
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated
with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan
extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

Cell viability measurement:
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

Number of replicates:

In this assay, two replicates for test item were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non specific OD evaluation.

Irritation / corrosion parameter:

% tissue viability

Value:

94.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
- Direct-MTT reduction: As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT.
- Colour interference with MTT: As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.006, Non Specific Colour % (NSCliving%) was calculated as 0.8%. This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the two negative control tissues was in the recommended range (0.826).
- Acceptance criteria met for positive control: The two positive control treated tissues showed 1.0% viability demonstrating the proper performance of the assay.
- Acceptance criteria met for variability between replicate measurements: The difference of viability between the two test item-treated tissue samples in the MTT assay was 8.9%. The difference of viability between the two negative control tissue samples in the MTT assay was 7.9 %.
- Range of historical values if different from the ones specified in the test guideline:N/A

Validity of the test:

After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the two negative control tissues was in the recommended range (0.826).

The two positive control treated tissues showed 1.0% viability demonstrating the proper performance of the assay.

The difference of viability between the two test item-treated tissue samples in the MTT assay was 8.9%.

The difference of viability between the two negative control tissue samples in the MTT assay was 7.9 %.

The mean OD value of the blank samples (acidified isopropanol) was 0.048.

All these parameters were within acceptable limits and therefore the study was considered to be valid.

Additional controls:

As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.006, Non Specific Colour % (NSCliving%) was calculated as 0.8% . This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro EPISKIN™(SM) model test with Sopromine 1686, the results indicate that the test item is non-corrosive to the skin.

Executive summary:

An in vitro skin corrosivity test of Sopromine 1686 test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Disks of EPISKINTM(SM) (two units) were treated with Sopromine 1686 test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified

spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control).

Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with Sopromine 1686, the mean cell viability was 94.3% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with Sopromine 1686 (Batch number: 3900075), the results indicate that the test item is non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-23 - 2018-03-20

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified (adult donors)

Justification for test system used:

The irritation potential of a chemical may be predicted by measurement of its cytotoxic effect, as reflected in the MTT assay, on the EPISKINTM (SM) reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD No. 404).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM (SM) (Manufacturer: SkinEthic, France)
- Tissue batch number(s): 8-EKIN-004
- Production date: 23 January 2018
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 24 January 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (25.4-26.0°C)
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: one washing with PBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Thermo Fisher Scientific
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 24072800, Serial
Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Quality control: EPISKINTM (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality contro experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM (SM) test kits used in the present study).
- Contamination: All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.

NUMBER OF REPLICATE TISSUES: Three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : two
- Method of calculation used: Non specific MTT reduction calculation (NSMTT%):
NSMTT (%) = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control killed tissues OD
ODKT: test item treated killed tissues OD
ODNC: negative control OD

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

PREDICTION MODEL / DECISION CRITERIA:
The test item considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The test item was applied as supplied, no formulation was required.

The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

Test Item
First an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then ~86.8 mg of the test item was applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip without damaging the epidermis. The amount was sufficient to cover the epidermal surface.
Note: Due to the physical properties of the test item, it was used as solid.

Negative and positive controls
50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (25.4-26.0°C).

Duration of post-treatment incubation (if applicable):

RINSING
After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

MTT test
After the 42 hours incubation, all EPISKINTM (SM) units (except the two living colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM (SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere.

Formazan extraction
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm)
for formazan extraction.

Cell viability measurements
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

Number of replicates:

In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation.

Irritation / corrosion parameter:

% tissue viability

Value:

94.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
- Direct-MTT reduction: Yellow colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.
- Colour interference with MTT: As the test item was coloured, two additional test item-treated living tissues were usednfor the non-specific OD evaluation. The mean optical density (measured at 570 nm) of
tissues was 0.009, Non Specific Colour % was calculated as 1.3%. Thisnvalue was below 5%, therefore additional data calculation was not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was in the recommended range (0.653). Standard deviation of the viability results for negative control samples
was 1.4%.
- Acceptance criteria met for positive control: The positive control treated tissues showed 4.1% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive
control samples was 0.4%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.3%.
- Range of historical values if different from the ones specified in the test guideline: N/A

ADDITIONAL CONTROLS

Yellow colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.009, Non Specific Colour % was calculated as 1.3%. This value was below 5%, therefore additional data calculation was not necessary.

VIABILITY RESULTS

The mean OD values for the test item treated skin samples showed 94.8% relative viability compared to the negative control.

VALIDITY OF THE TEST

After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the three negative control tissues was in the recommended range (0.653). Standard deviation of the viability results for negative control samples was 1.4%.

The positive control treated tissues showed 4.1% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.4%.

The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.3%.

The mean OD value of the blank samples (acidified isopropanol) was 0.046.

All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Following exposure with Sopromine 1686, the mean cell viability was 94.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro EPISKINTM (SM) model test with Sopromine 1686, the results indicate that the test item is non-irritant to skin, UN GHS Classification: No Category (since the test item is known not to be corrosive).

Executive summary:

An in vitro skin irritation test of Sopromine 1686 test item was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a >95% humidified atmosphere.

The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere, protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with Sopromine 1686, the mean cell viability was 94.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKINTM (SM) model test with Sopromine 1686 (Batch number: 3900075), the results indicate that the test item is non-irritant to skin, UN GHS Classification: No Category (since the test item is known not to be corrosive).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-10 - 2018-05-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint-Pierre-sur-Dives, France.
- Number of animals: unknown
- Characteristics of donor animals (e.g. age, sex, weight): bovine cattle were up to 12 months old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): the eyes were immerged in containers filled with cooled buffered Hanks medium and placed into a cooling-box with a sufficient amount of ice packs to ensure cooling until arrival at Citoxlab France. Containers with smooth internal surfaces were used for the transport to avoid damage to the corneas. Hank’s medium contained an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].
Upon arrival at Citoxlab France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.
Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared. The corneas were then used immediately.
- indication of any existing defects or lesions in ocular tissue samples: Any eyes with defects were discarded.
- Indication of any antibiotics used: penicillin/streptomycin

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 mg (± 75 mg)
- Concentration (if solution): as such

Duration of treatment / exposure:

10 minutes (± 30 seconds)

Duration of post- treatment incubation (in vitro):

2 hours (± 10 minutes)

Number of animals or in vitro replicates:

The test item, the negative and the positive control were tested on three corneas each.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
The corneas were then used immediately.
The corneas were mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber. Each cornea was identified with the corresponding holder number.
For pre-incubation, both chambers of the corneal holder were filled to overflowing with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) at room temperature. The posterior chamber was always filled first to maintain the natural concave shape of the cornea.
After making sure that no air bubbles were present within the holder, it was immersed in a water bath, horizontally (cornea positioned vertically), up to approximately three quarters of its height. The holders were pre incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C (± 1°C).
At the end of the pre-incubation period, the medium was removed from both chambers of the holder using a metal gavage tube attached to a vacuum pump to ensure complete evacuation. They were refilled with fresh cMEM without phenol red (previously heated to +32°C), starting with the posterior chamber and taking care that no air bubbles were present. The chambers were re-sealed and the corneas were examined macroscopically through the holder to detect the presence of any defects. Then, the opacity of the cornea was measured to obtain OPT0 (see § Opacity measurements).
Corneas that showed any macroscopic defect or an OPT0 value over 7 were discarded.

QUALITY CHECK OF THE ISOLATED CORNEAS:
The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded.

NUMBER OF REPLICATES:
The test item, the negative and the positive control were tested on three corneas each.

NEGATIVE CONTROL USED: 0.9% Sodium Chloride (NaCl)

SOLVENT CONTROL USED (if applicable): N/A

POSITIVE CONTROL USED: Absolute Ethanol

APPLICATION DOSE AND EXPOSURE TIME: 750 mg (± 75 mg) for 10 minutes (± 30 seconds)

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: four times.
the corneas were rinsed three times with pre-warmed cMEM containing phenol red (i.e. until the test item had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

- POST-EXPOSURE INCUBATION: 2 hours (± 10 minutes)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.

- Corneal permeability: After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution.
As the test item is a non-surfactant liquid, the concentration of the fluorescein solution was 4 mg/mL.
Before use, the fluorescein solution was validated. For this purpose, the solution of fluorescein was diluted in cMEM in order to obtain a 5 μg/mL solution and the Optical Density at a wavelength of 490 nm (OD490 nm) of this dilution was measured. As the value obtained was between 0.850 and 0.940, the fluorescein solution was validated.
For each series of three corneas, a chronometer started from the fluorescein solution application time of the first cornea of the series. The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes).
At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank).

- Others (e.g, pertinent visual observations, histopathology): After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: YES

Irritation parameter:

in vitro irritation score

Value:

ca. 0

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 notable opaque spots or irregularities were observed on negative control and test item-treated corneas.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: no

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item, Sopromine 1686, was identified as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item,SOPROMINE 1686, to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The design of this study was based on the OECD Guideline 437 and the study was performedin compliance with Citoxlab France standard operating proceduresand with the OECD Principles of Good Laboratory Practice.

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.

A single experiment was performed using three corneas for each treated series (test item, positive control and negative control).

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item, applied undiluted, was evaluated in a single experiment using a treatment time of 10 minutes and the open-chamber treatment method. Negative and positive controls were applied using the same treatment time but using the closed-chamber treatment method.

At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.

The corneas were then incubated for 2 hours (± 10 minutes)at +before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes(± 5 minutes)at +32°C.

At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

No notable opaque spots or irregularities were observed on each test item-treated cornea.

All acceptance criteria were fulfilled. The study was therefore considered as valid.

The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0.

As the mean IVIS was ≤ 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Under the experimental conditions of this study, the test item,SOPROMINE 1686,was identified asnot requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two Klimisch score 1 studies are available to assess the skin corrosive and irritating potential and were used as key studies study:

In the first one, an in vitro skin corrosivity test of Sopromine 1686 test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline. Disks of EPISKINTM(SM) (two units) were treated with Sopromine 1686 test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin. Following exposure with Sopromine 1686, the mean cell viability was 94.3% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EPISKIN™(SM) model test with Sopromine 1686 (Batch number: 3900075), the results indicate that the test item is non-corrosive to the skin.

In the second study, an in vitro skin irritation test of Sopromine 1686 test item was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline. Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a >95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere, protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin. Following exposure with Sopromine 1686, the mean cell viability was 94.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EPISKINTM (SM) model test with Sopromine 1686 (Batch number: 3900075), the results indicate that the test item is non-irritant to skin, UN GHS Classification: No Category (since the test item is known not to be corrosive).

One Klimisch score 1 study is available to assess the eye corrosive and irritating potential of the registered substance and was used as key study:

In this study, performed according to OECD guideline no. 437 (BCOP test) and in compliance with GLPs, Sopromine 1686 was applied for 10 minutes as it is on isolated bovine corneas. No notable opaque spots or irregularities were observed on each test item-treated cornea. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0.

As the mean IVIS was ≤ 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage

Justification for classification or non-classification

In an in vitro skin corrosion (OECD 431) and in vitro skin irritation (OECD 439) tests performed using a reconstructed human epidermis model,

the mean relative cell viability following exposure with the registered substance was above the threshold of 35% and 50% compared to the negative control, respectively, indicating that the substance is not corrosive nor irritant to skin.

In addition, in a Bovine Corneal Opacity and Permeability test (OECD 437), the mean In Vitro Irritancy Score was equal to 0 indicating that the substance is not corrosive nor irritant to eyes.

In conclusion the registered substance is not classified for skin and eye irritation/corrosion according to CLP and GHS-UN regulations.