Ethyl 2-ethylhexanoate

Endpoint summary

Description of key information

Skin irriation/corrosion: One in vitro study for skin corrosion, conducted in 2017 under GLP and following OECD 431 and one in vitro study for skin irritation, conducted in 2017 under GLP and following OECD 439 are available. Accoding to the results, the test substance was identified as non-corrosive and non-irritant to the skin.

Eye irritation: Two in vitro studies for eye irritation are available. The test substance could not be classified according to one study conducted in 2017 under GLP following OECD 437. However the test substance was considered as producing eye irritation/ reversible effects on the eye (CLP Category 2) or serious eye damage/ irreversible effects on the eye (CLP Category 1) according to the other study conducted in 2017 under GLP following OECD 492.

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:

From 24/10/2016 to 04/05/2017

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

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: Reconstructed epidermis of normal human keratinocytes

Cell source:

other: Adult donors

Vehicle:

unchanged (no vehicle)

Details on test system:

-Test System
Commercial Name: EPISKIN™ - 0.38 cm2
Supplier: SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
Batch: 16EKIN049
Arrived at RTC on: 06 December 2016

-Preparation and pre-treatment incubation period
The test system was shipped onMonday and received on Tuesday. According to the supplier procedure, tissues were prepared as follows:
Alive tissues: at arrival, plates were opened under a sterile airflow and each insert,containing the epidermal tissue, was carefully taken out and placed in a 12-well plate in which each well had previously been filled with 2 mL/well SkinEthicMaintenance Medium.
Culture plates were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.

-Media
Maintenance Medium: SkinEthic; batch: 16MAIN3 080
AssayMedium: SkinEthic; batches: 16ESSC 048 and 16ESSC 052

-Preliminary test
Direct MTT reduction test (Step 1)
Non-specific reduction of MTT was evaluated as follows: two mL of MTT ready-to-use solution (0.3 mg/mL) was incubated with 20 µL of test item at 37°C, 5% CO2 and saturated humidity for 3 hours, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.

Colouring potential test (Step 2)
Chemicals’ colouring potential was assessed for potential interaction with the test system. 20 µL of the test item was added to 180 µL of distilled water (Baxter; batch no. 15I030I) in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes. At the end of the incubation time, colouring of the solution/suspension evident to the unaided eye and measured by spectral analysis at 595 nm, was evaluated.

-Main Assay
Treatment
In theMain Assay, alive tissues were treated with the test item, positive and negative controls.

Exposure period
An exposure time of 15±0.5 minutes was allowed in a ventilated cabinet at room temperature.

Washing
At the end of the exposure, each tissue was rinsed with approximately 25mL of sterile D-PBS, filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2mL/well of maintenance medium.

Post-exposure period
A 42 ± 1 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.

MTT staining
Each tissue insert was incubated with 2 mL/well of MTT ready-to-use solution, with the exception of tissues used for the unspecific colouring potential control. Plates were incubated for approximately 3 hours at 37°C, 5% CO2 and saturated humidity. At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions. The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 µL of acidic isopropanol. Tubes were preserved for approximately 3 days at 4°C to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (14000 rpm for 2 minutes) and aliquots of 200 µL from each sample were read in duplicate for their absorbance at 595 nm. Optical Density (OD) values were recorded. Six aliquots (200 µL) of acidic isopropanol were analysed and used as blank. In order to ensure the spectrophotometer linear range, an MTT formazan calibration curve was performed.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

20 µl for negative control, positive control and test item

Duration of treatment / exposure:

An exposure time of 15±0.5 minutes was allowed

Duration of post-treatment incubation (if applicable):

A 42 ± 1 hour recovery period was allowed

Number of replicates:

3 replicates for negative control, positive control and test item, 2 replicates for only test item without MTT.

Irritation / corrosion parameter:

other: other: cell viability in % (obtained from optical density)

Value:

74

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™.
The blank, negative and positive controls gave acceptable results and the study was accepted as valid.
The mean cell viability of the test item treated tissues, after the blank subtraction, was 74%.
Based on the results obtained, the test item is classified as not irritant to the skin (UN GHS No Category).

Executive summary:

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system, being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor. Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducingMTT per se. No relevant interaction between the test teim andMTT was observed. In a second step, the test item was assayed for the ability of colouring water per se. A colourless solution was observed; spectral analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 0.604. This unexpected result indicated that the test item could have a potential interfering ability, thus an additional control was added in theMain Assay.

In theMain Assay, the test item was applied as supplied in three replicates at the treatment level of 20 µL/epidermis unit, each measuring 0.3 cm2 (treatment level: 53 µL/cm2). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µL/epidermis unit. In order to verify if the test item results had to be corrected, the non specific colour (NSCliving) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control.

In the Main Assay, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the negative control mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

The positive control caused the expected cell death (5% of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.5). Based on the stated criteria (mean viability ≤ 40% and SD of % viability ≤ 18), the assay was regarded as valid.

The NSCliving was 5%, thus only the OD-blank background subtraction was performed.

The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 74% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 14.7 (lower than 18, as stated in the Study Protocol).

Based on the results obtained, the test item is classified as not irritant to the skin.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 12/10/2016 to 04/05/2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: Reconstructed epidermis of normal human keratinocytes

Cell source:

other: Adult donors

Vehicle:

unchanged (no vehicle)

Details on test system:

Test System
-EPISKIN™
Commercial Name EPISKIN™ - 0.38 cm2
Supplier SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
Batch 16-EKIN-042
Arrived at RTC on 19 October 2016

-Preparation and pre-treatment incubation period
The test system was shipped on Monday and received on Wednesday. According to the supplier procedure, at arrival, plates were opened under a sterile airflow and each insert, containing the epidermal tissue, was carefully taken out and placed in a 12-well plate in which each well had previously been filled with 2mL/well SkinEthicMaintenanceMedium. Culture plates were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.

-Media
MaintenanceMedium SkinEthic; batch: 16-MAIN3-071
AssayMedium SkinEthic; batches: 16 ESSC 044 and 16 ESSC 045

-Preliminary test
Direct MTT reduction test (Step 1)
Non-specific reduction of MTT was evaluated as follows: two mL of MTT ready-to-use solution (0.3mg/mL) was incubated with 50 µL of test item at 37°C, 5% CO2 and saturated humidity for 3 hours, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.

Colouring potential test (Step 2)
Chemicals’ colouring potential was assessed for potential interaction with the test system. 10 µL of test item was added to 90 µL of distilled water (Baxter; batch no. 15D2902) in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes. Colouring of the solution/suspension at the end of the incubation time was evaluated by spectral analysis at 595 nm.

-Main Assay
Treatment
InMain Assay, alive tissues were treated with the test item, positive and negative controls.

Washing
At the end of the exposure, each tissue was rinsed with approximately 25mL of sterile PBS, filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2mL/well of maintenance medium.

MTT staining
Each tissue insert was incubated with 2mL/well of MTT ready-to-use solution. Plates were incubated for 3 hours ± 5 minutes at 37°C, 5% CO2 and saturated humidity. At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions. The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 µL of acidic isopropanol. Tubes were mixed by vortexing and preserved overnight at room temperature to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (14000 rpm for 2 minutes) and aliquots of 200 µL from each sample were read in duplicate for their absorbance at 595 nm. Six aliquots (200 µL) of acidic isopropanol were analysed and used as blank. On the day of spectrophotometer analysis, quality control solutions of MTT formazan were prepared and analysed in order to ensure that the MTT formazan calibration curve was still appropriate.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL for negative control, positive control and test item

Duration of treatment / exposure:

Exposure times of 3, 60±5 and 240±5 minutes were allowed in a ventilated cabinet at room temperature.

Number of replicates:

2 for negative control, positive control and test item

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Treatment time: 3 minutes

Value:

111

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Treatment time: 60 minutes

Value:

101

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Treatment time: 240 minutes

Value:

119

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

-Preliminary test
Before theMain Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. No interaction was recorded between the test item and MTT in the test conditions. Thus no additional controls were added in the main phase for the evaluation of MTT non specific reduction. No precipitate was noted. In a second step, the test item was assayed for the ability of colouring water per se. No colouring potential of the test item in contact with water was recorded, with an OD value of 0.073. Therefore, no additional controls were added in the main phase for the evaluation of non specific colouring potential.

-Main Assay
A Main Assay was performed. The mean Optical Density of Blank Controls was 0.038, lower than the maximum acceptable value (0.1). All negative control mean OD values gave the expected baseline value and variability, in agreement with guideline indications. According to the method, each negative control mean value is considered the baseline value for the concurrent treatment series, thus they represent 100% of cell viability. Positive control results indicated an appropriate cell death with an acceptable relative cell viability (1.3% of the negative control value). Based on the stated criteria, the study was accepted as valid. The test item did not induce cell death in any replicate, at any treatment time.Intra-replicate variability was acceptable with a difference of viability between the two replicates lower than 30%, for all treatment times. Based on the results obtained, the test item IROTYL is identified as non-corrosive to the skin.

Interpretation of results:

GHS criteria not met

Conclusions:

The potential of the test item to be corrosive to the skin was investigated through an in vitro skin corrosion study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™.
The blank, negative and positive controls gave acceptable results, at all treatment times, thus the study was accepted as valid. The mean cell viability of the test item treated tissues, after the blank subtraction, was higher than 35% at all treatment times. Based on the results obtained, the test item IROTYL is identified as non-corrosive to the skin.

Executive summary:

The potential of the test item to be corrosive to the skin was investigated through an in vitro skin corrosion study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 431. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 3, 60 and 240 minutes. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system, being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor.

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In particular, the test item was assayed for the ability of reducing MTT and colouring water per se. No interaction was recorded between the test item and MTT in test conditions similar to those of theMain Assay. Moreover, no colouring potential of the test item in contact with water was recorded, with an OD value of 0.073. Based on these results, no additional controls were added in the main phase.

In the Main Assay, for each treatment time, the test item was applied as supplied in two replicates at the treatment level of 50 µL/epidermis unit, each measuring 0.38cm2 (treatment level: 131.6 µL/cm2). Positive and negative controls (Glacial acetic acid and Physiological saline, respectively) were concurrently tested, in the same number of replicates and test conditions at the treatment level of 50 µL/epidermis unit. Positive control was included only at the longest treatment time of 240 minutes, while a negative control was included for each treatment time.

In theMain Assay, the negative controls gave the expected baseline value (Optical Density values ≥ 0.6 and ≥ 1.5) and variability (difference of viability between the two replicates lower than 30%), at each treatment time, in agreement with the guideline indications. For each treatment time, the concurrent negative control mean value is considered the baseline value of the treatment series and thus represents 100% of cell viability.

The positive control caused the expected cell death (1.3% of cell viability, when compared to the negative control).

Based on the stated criteria, the assay was regarded as valid.

The test item did not induce cell death in any replicate, at any treatment time. Each mean cell viability, after the concurrent blank subtraction, was as follows:

Treatment time (minutes) Mean cell viability (%)

3                                          111

60                                       101

240                                     119

Intra-replicate variability was acceptable with a difference of viability between the two replicates lower than 30%, for all treatment times.

Based on the results obtained, the test item IROTYL is identified as non-corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

weight of evidence

Study period:

From 16/2/2017 to 26/4/2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

-Commercially available EpiOcularTM kit.
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

-Origin
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Day of delivery: 28: Mar. 2017
Batch no.: 23773

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 μL of the controls and the test item were applied in 1 minute intervals.

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Duplicate

Details on study design:

Pre-Tests
-Assessment of Direct Reduction of MTT by the Test Item
The test item Irotyl was tested for the ability of direct formazan reduction. To test for this ability, 50 μL of the liquid test item were added to 1 mL of MTT reagent in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 for 3 hours. 1 mL of MTT reagent plus 50 μL of H2O demin. was used as negative control. The MTT reagent did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.

-Assessment of Coloured or Staining Test Items
The test item is colourless. To assess, whether the test item will become coloured after contact with water or isopropanol, 50 μL of the liquid test item were added to 1 mL of sterile demin. water in a 6-well plate and incubated in the dark at 37 ± 1 °C, 5 ± 1 % CO2 for 1 hour. After incubation, no colour development was visible. Therefore, the main test was performed without colourant controls.

Main Test
-Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the MTT solvent directly before use. The assay medium was warmed in the water bath to 37 ± 1°C. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 –100 % relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours and 30 minutes.

-Exposition and Post-Treatment
After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and then incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes. After that, 50 μL of the controls and the test item were applied in duplicate in 1 minute intervals. This was done in such a fashion that the upper surface of the tissue was covered. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of the exposure time, the inserts were removed from the plates in 1 minute intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred to and immersed in 5 mL of pre-warmed assay medium in a pre-labelled 12-well plate for 12 minutes resp. 14 minutes post soak at room temperature. After that, each insert was removed from the medium, the medium was decanted off the tissue and the insert was blotted on absorbent material and transferred into the respective well of a pre-labelled 6-well plate containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 120 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100% relative humidity. After the post-treatment incubation, the MTT Assay was performed.

-MTT Assay and Extraction
A 24-well-plate was prepared with 300 μL freshly prepared MTT-reagent in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into the empty 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The
plate was sealed and stored in the refrigerator overnight. On the next day, the plate was shaken for 2 hours at room temperature, protected from light.

-Measurement
The inserts were pierced with an injection needle, taking care that all colour is extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate which was read in a plate spectrophotometer at 570 nm.

Irritation parameter:

in vitro irritation score

Value:

27.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

other: the test substance is considered as producing eye irritation/reversible effects on the eye (CLP Category 2) or serious eye damage/irreversible effects on the eye (CLP Category 1)

Conclusions:

Under the conditions of this test, the test substance is considered as producing eye irritation/reversible effects on the eye (CLP Category 2) or serious eye damage/irreversible effects on the eye (CLP Category 1).

Executive summary:

The test item Irotyl was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 30 minutes. 50 μL of the liquid test item was applied to each tissue. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control, methyl acetate was used as positive control.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.5. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 26.6 % (< 50%). Variation within tissue replicates was acceptable (≤ 20%). After treatment with the test item, the mean value of tissue viability was 27.7 %. This value is well below the threshold for eye irritation potential (≤ 60%). Under the conditions of this test, Irotyl is considered as producing eye irritation/ reversible effects on the eye (CLP Category 2) or serious eye damage/ irreversible effects on the eye (CLP Category 1).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 21-10-2016 to 14-03-2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

other: Bos primigenius Taurus (fresh bovine corneas)

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 h.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The respective substance (negative control solution, test item or positive control) was applied by pipetting 750 μL of the appropriate liquid through the refill hole in the holder on the cornea.

Duration of treatment / exposure:

Exposure time on the corneas was 10 minutes at 32 ± 1 °C.

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 hours at 32 ± 1 °C (post-incubation).

Number of animals or in vitro replicates:

For each treatment group (negative control solution, test item and positive control), three replicates were used.

Details on study design:

-Preparations
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C. On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate. After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

-Experimental Parameters
Date of treatment: 26. Oct. 2016
Incubation time: 10 min.
Negative control: HBSS
Positive control: Dimethylformamide (undiluted)

-Method Description
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative control solution resp. test item resp. positive control were applied to each replicate. According to the characteristics of the test item, the following treatment procedure was performed:

The “closed chamber-method” is used for liquid substances. The respective substance (negative control solution, test item or positive control) was applied by pipetting 750 μL of the appropriate liquid through the refill hole in the holder on the cornea. The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with the test item. Exposure time on the corneas was 10 minutes at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 hours at 32 ± 1 °C (post-incubation). After the post-incubation, the cMEM without phenol red was renewed in both chambers. Then, the final opacity value of each cornea was recorded. The cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution (concentration: 4 mg/mL) was added to the front chamber. The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the liquid was measured with the spectrophotometer at 492 nm.

Irritation parameter:

in vitro irritation score

Value:

9.61

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS-solution	0,82	0,04	1800,92%
	-0,16
	-0,54
Test Item	8,67	9,61	9,81
	10,56
	9,61
Positive Control DMF undiluted	121,75	135,11	10,61
	150,26
	133,33

Interpretation of results:

study cannot be used for classification

Conclusions:

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category.

Executive summary:

One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old. The test item Irotyl was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured. Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 135.11. Under the conditions of this study, the test item Irotyl showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 9.61.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation/corrosion:

Based on the study reports, the test substance does not show corrosion nor irritation potential. Scores are clearly below the thresholds defined in GHS and in CLP to classify one substance as skin irritation/corrosion. Therefore the registered substance is considered to be not classified for skin irritation/corrosion.

 

Eye irritation:

Based on the study following OECD 492, the test substance shows irritation potential and is considered as producing eye irritation/ reversible effects on the eye (CLP Category 2) or serious eye damage/ irreversible effects on the eye (CLP Category 1). The scores are as strong as the one of the positive control, Methyl Acetate (Eye irritation: Category 2 by Harmonised Classification). Because this test method does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), further testing would be needed to categorize test substance (1).

 

Based on the study following OECD 437, the test substance shows very weak irritation potential and the irritation score is way below the threshold for Category 1 (test result IVIS Score = 9.6). As shown in the validation study of this test method conducted with 158 chemicals with replicates in up to 12 laboratories (2), all liquids with an IVIS Score < 10 have a consensus in-vivo GHS Classification of Category 2A (reversible effects on the eye/irritating to the eyes), Category 2B (reversible effects on the eye/mildly irritating to the eyes) or Non Irritant (not classified for eye irritation).

 Therefore it could be assumed from the test result that test substance does not possess the potential for serious eye damage/irreversible effects on the eye.

 

Combining these findings, and considering the registered substance shows a very low irritant response in the skin and a mild pH in water (4,4), it is concluded that the registered substance is considered to be classified as eye irritation/reversible effects on the eye (Category 2) (3) & (4).

 

(1) OECD. (2015) Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. OECD Guideline for Testing of Chemicals No. 492. Organisation for Economic Co-operation and Development, Paris.

 

(2) See attached document "Appendix D2 Comparison of In Vivo and In Vitro Ocular Irritancy Classifications" of the document "ICCVAM. 2006a. Current Status of In Vitro Test Methods for Identifying Ocular Corrosives and Severe Irritants: Bovine Corneal Opacity and Permeability Test Method. NIH Publication No.: 06-4512. Research Triangle Park: National Toxicology Program".

 

(3) Scott L, Eskes C, Hoffmann S, Adriaens E, Alepè N, Bufo M, Clothier R, Facchini D, Faller C, Guest R, Harbell J, Hartung T, Kamp H, Varlet BL, Meloni M, McNamee P, Osborne R, Pape W, Pfannenbecker U, Prinsen M, Seaman C, Spielmann H, Stokes W, Trouba K, Berghe CV, Goethem FV, Vassallo M, Vinardell P, Zuang V. "A Proposed eye irritation testing strategy to reduce and replace in vivo studies using Bottom-Up and Top-Down approchaes"  Toxicol In vitro, 2010, 24 (1), 1 -9

 

(4) Ramirez, T.; Kolle, S. N.; Schulte, S.; Schulz, M.; Mehling, A.; Kleber, M.; Teubner, W.; Landsiedel, R. “Toxicological Testing of Cosmetic Ingredients Using Alternative Methods – The Supplier’s Perspective”. IFSCC MAGAZINE; 2013, 16(4):263-275 ; Pub: Germany, International Federation of Societies of Cosmetic Chemists, 2013