Registration Dossier

Administrative data

Description of key information

Skin corrosion in vitro: An analogue test item was considered to be non-corrosive to skin because mean cell viability compared to the negative control was determined to be 79.0 % after 4 hours using the EPISKIN (SM) model (OECD 431).

Skin irritation in vitro: An analogue test item was considered to be irritant to skin because mean viability of the test item treated tissues was determined to be 6.6 % compared to the negative control after the 15-minute exposure period and 42-hours post-exposure incubation using the EPISKIN (SM) model (OECD 439).

 

Skin irritation in vivo: An analogue test item was determined to be irritant to skin because, although mean scores from 24, 48 and 72-hour observations were zero for oedema and 2.3 for erythema in all three animals, the observed erythema did not reverse over a period of 14 days (OECD 404, EU Method B.4 and OPPTS 870.2500).

Eye damage/irritation in vitro: An analogue test item was not classified as a severe irritant and not classified as non-irritant using the in vitro isolated chicken eye test method (OECD 438). It was therefore concluded that an in vivo study was required.

 

Eye damage/irritation in vivo: An analogue test item, applied to rabbit eye mucosa, caused significant conjunctival and corneal irritant effects (OECD 405). Ocular effects were declared irreversible after veterinary consultation and the animal was euthanised for reasons of animal welfare. No further test was deemed necessary.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 October 2016 to 07 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
other: adult
Justification for test system used:
INTRODUCTION
- 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.
- The present test is based on the experience that corrosive chemicals, formulations, products or mixtures show cytotoxic effects following short-term exposure of the stratum corneum of the epidermis. The purpose of this study is to predict the skin corrosivity potential of a chemical by assessment of its effect on a reconstituted human
epidermis.
- The EPISKINTM(SM) is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin corrosivity testing involves the topical application of test materials to the surface of the skin, and the subsequent assessment of their effects on cell viability. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT (Fentem et al., 1998). The test can be used for classification as not corrosive, or as 1A, 1B or 1C (COMMISSION REGULATION (EU) 2016/863 of 31 May 2016).
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
- Human skin: EPISKIN (SM) (Manufacturer: SkinEthic, France, Batch No.: 16-EKIN-040, Expiry Date: 10 October 2016) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
- Quality control: EPISKIN (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkin (SM) Test Kits used in the present study) and are documented in Appendix 2 (attached).
- Justification for selection of the test system: The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

KIT CONTENTS
- Units: EPISKIN (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
- Plate: 12-well assay plate
- Punch: EPISKIN (SM) biopsy punch for easy sampling of epidermis.
- Medium: Flask of sterile “Maintenance Medium” (Batch No.: 16 MAIN3 067; Exp. Date: 12 October 2016); Flask of sterile “Assay Medium” (Batch No.: 16 ESSC 043; Exp. Date: 12 October 2016).

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

KIT RECEPTION
- In each case, the pH of the agar medium used for transport was checked by checking the colour of the medium (orange colour = good; yellow or violet colour = not acceptable)
- The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40 °C (the colour change is irreversible, independent of the length of the period above 40 °C; white colour = good; grey or black colour = not acceptable)
- The kits were found to be in good order at reception.

STORAGE
- The EPISKIN (SM) kits were kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2 to 8 °C until the initiation of the test.

ADDITIONAL MATERIALS
- Details of chemicals used in the performed experiments are summarised in the table attached.
- MTT solution: MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 05 October 2016) was stored in refrigerator (2 to 8 °C) protected from light. It was diluted with pre-warmed (37 °C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.
- Acidified isopropanol: Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl
(1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

INDICATOR FOR POTENTIAL FALSE VIABILITY
- Chemical action by the test material on MTT may mimic that of cellular metabolism leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.
- If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement.
- Approximately 50 μl of test item was added to 2 mL MTT working solution and mixed.
The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 3 hours and then any colour change was observed (test items which do not interact with MTT: yellow; test items interacting with MTT: blue or purple).
- After three hours incubation, yellow colour in the mixture was detected; therefore additional controls were not necessary in the experiment.
- Prior to treatment, the test item was evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). As the test item had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSC living %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues.
- Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.

PRE-INCUBATION (Day -1)
- 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.

APPLICATION (Day 0)
- 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 the test item or negative control or positive control substance in experiment.
- Test item (50 μL) was applied evenly to the epidermal surface of each of two test units.
- Physiological saline (50 μL) was added to each of the two negative control skin units.
- Glacial acetic acid (50 μL) was added to each of the two positive control skin units.
- The plates with the treated epidermis units were incubated for 4 hours (± 10 min) at room temperature (22.7 to 23.9 °C) covered with the plate lids.

RINSING (Day 0)
- After the incubation times, all test item or positive control treated tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface.
- The negative control tissues were also processed accordingly.
- The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MTT TEST (Day 0)
- 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 colour control units).
- The lid was replaced and the plate incubated at 37 °C in an incubator with 5% CO2 for 3 hours, protected from light.

FORMAZAN EXTRACTION (Day 0)
- 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 (approximately 150 rpm) for formazan extraction.
- A blank sample containing 2 mL of acidified isopropanol was processed in parallel.
- Note: Before the epidermis was cut from each skin unit, white residue test item was found. However the remaining test item fell off after the epidermis was cut from each skin unit.

CELL VIABILITY MEASUREMENTS (Day 1)
- Following the formazan extraction, 2 × 200 μL samples 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.
- The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, 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.

CALCULATION OF VIABILITY PERCENTAGES
- The data calculation using two replicates is shown below. Results were calculated in a similar way when more replicates are used.

BLANK
- The mean of the 6 blank OD values was calculated.

NEGATIVE CONTROL
- Individual negative control OD values (NC raw) were corrected with the mean blank OD using the equation OD Negative Control (OD NC) = OD NC raw – OD blank mean.
- The corrected mean OD of the 2 negative control values was also calculated: this corresponds to 100% viability.

POSITIVE CONTROL
- Individual positive control OD values (PC raw) were corrected with the mean blank OD using the equation OD Positive Control (OD PC) = OD PC raw – OD blank mean.
- The corrected mean OD of the 2 positive control values was calculated.
- The % viability for each positive control replicate was calculated relative to the mean negative control using the equations Positive Control 1 % = (OD PC1 / Mean OD NC) x 100 and Positive Control 2 % = (OD PC2 / Mean OD NC) x 100.
- The mean value of the 2 individual viability % for positive control was calculated using the equation Mean PC % = (PC1 % + PC2 %) / 2.

TEST ITEM
- Individual test item OD values (TT raw) were corrected with the mean blank OD using the equation OD Treated Tissue (OD TT) = OD TTraw – OD blank mean.
- The corrected mean OD of the 2 test item values was calculated.
- The % viability for each test item replicate was calculated relative to the mean negative control using the equations Treated Tissue 1 % = (OD TT1 / Mean OD NC) x 100 and Treated Tissue 2 % = (OD TT2 / Mean OD NC) x 100.
- The mean value of the 2 individual viability % for test item was calculated using the equation Mean TT % = (TT1 % + TT2 %) / 2.
- Variability for 2 disks = (Disk 1 - Disk 2) / ((Disk 1 + Disk 2) / 2) x 100 %.

DATA CALCULATIONS FOR TEST ITEMS HAVING MTT-INTERACTING POTENTIAL
- Test items that interfere with MTT can produce non-specific reduction of the MTT. In this case, additional control samples were used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value was corrected by the result of the additional controls before calculation of viability% using the equation Non specific MTT reduction calculation (NSMTT%) = [(OD KT – OD KNC) / OD NC] x 100 where OD KNC = negative control treated killed tissues OD; OD KT = test item treated killed tissues OD; OD NC = negative control OD.
- If NSMTT% is ≤ 50%, then true MTT metabolic conversion (TOD TT) had to be undertaken using the equation TOD TT = [OD TT – (OD KT – OD KNC)] where OD TT = test item treated viable tissues.
- The % relative viability (RV%) for each test item replicate was calculated relative
to the mean negative control using the equations RV1 % = [TOD TT1 / Mean OD NC] x 100 and RV2 % = [TOD TT2 / Mean OD NC] x 100.
- The mean value of the two individual relative viability % results for test item was calculated using the equation Mean Relative Viability % = (RV1 % + RV2 %) / 2.
- If NSMTT% is > 50% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

DATA CALCULATION FOR TEST ITEMS HAVING COLOURING POTENTIAL
- For test items detected as able to stain the tissues the non-specific OD was evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability % using the equation Non Specific Colour % (NSC living %) = (Mean OD CTV / Mean OD NC) x 100 where OD CTV = test item treated viable tissue (not incubated with MTT); OD NC = negative control OD (incubated with MTT).
- If NSC living % is ≤ 5 % then the normal calculation mode was used.
- If NSC living % is > 5 % and ≤ 50 %, then additional correction (TOD TT) had to be undertaken using the equation TOD TT = [OD TV – OD CTV] where ODTT = test item treated viable tissue (incubated with MTT); OD CTV = test item treated viable tissue (not incubated with MTT).
- The % relative viability (RV %) for each test item replicate was calculated relative to the mean negative control using the equations RV1 % = [TOD TT1 / Mean OD NC] x 100 and RV2 % = [TOD TT2 / Mean OD NC] x 100.
- The mean value of the two individual relative viability % results for test item was calculated using the equation Mean Relative Viability % = (RV1 % + RV2 %) / 2.
- If NSMTT% is > 50% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

DATA CALCULATION FOR TEST ITEMS HAVING BOTH MTT-INTERACTING AND COLOURING POTENTIAL
- Test items detected as able to both stain the tissues and interfere with MTT may also require a third set of controls before calculation of the “true” viability %.
- Non Specific Colour % with killed tissues (NSC killed %) = (Mean OD CTK / Mean OD NC) x 100 where OD CTK = test item treated killed tissues (not incubated with MTT); OD NC = negative control OD (incubated with MTT) and TOD TT = [OD TT – (OD KT – OD KNC) – Mean OD CTV + Mean OD CTK] where OD TT = test item treated viable tissues (incubated with MTT); OD KT = test item treated killed tissues OD; OD KNC = negative control killed tissues OD; OD CTV = test item treated viable tissues (not incubated with MTT); OD CTK = test item treated killed tissues (not incubated with MTT).
- The % relative viability (RV %) for each test item replicate was calculated relative to the mean negative control using the equations RV1 % = [TOD TT1 / Mean OD NC] x 100 and RV2 % = [TOD TT2 / Mean OD NC] x 100.
- The mean value of the two individual relative viability % results for test item was calculated using the equation Mean Relative Viability % = (RV1 % + RV2 %) / 2.

VALIDITY OF THE TEST
- The mean OD value of the two negative control tissues should be ≥ 0.6 and ≤ 1.5 and
negative control OD values should not be below historically established boundaries.
- The acceptable mean viability % range for positive control is ≤ 20 %.
- The difference of viability between the two tissue replicates should not exceed 30 %.
- The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.

INTERPRETATION OF TEST RESULTS
- The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with OECD No. 431 (OECD, 2016). 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).
- For more than 2 disks: If the mean value is ≥ 35 % and the variability is less than 50 % = Non Corrosive; if the mean value is < 35% and the variability is less than 50% = Corrosive.
- If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35 %. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 μL
Duration of treatment / exposure:
4 hours
Number of replicates:
Two
Irritation / corrosion parameter:
% tissue viability
Value:
79
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ADDITIONAL CONTROLS
- 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 0.005.
- The Non Specific Colour % (NSC living %) was calculated as 0.6% (see Table 1, attached). This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

VIABILITY RESULTS
- The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 2 (attached).
- The mean OD value for the test item treated skin samples showed 79.0 % relative viability.

VALIDITY OF THE TEST
- After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions in each case.
- The mean OD value of the two negative control tissues was in the recommended range (0.974).
- The positive control treated tissues showed 0.7% 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 7.1%.
- The difference of viability between the two negative control tissue samples in the MTT assay was 4.8%.
- The mean OD value of the blank samples (acidified isopropanol) was 0.045.
- All these parameters were within acceptable limits and therefore the study was considered to be valid.
- Historical control data are presented in Appendix 3 (attached).
Interpretation of results:
GHS criteria not met
Conclusions:
Mean cell viability was 79.0% compared to the negative control (after adjustment for non-specific MTT reduction) and the test item is considered to be non-corrosive to skin.
Executive summary:

GUIDELINE

An in vitro skin corrosivity test of test item was performed in a reconstructed human epidermis model. EPISKIN (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.

 

METHODS

Disks of EPISKIN (SM) (two units) were treated with 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 (NSC living) 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.

 

RESULTS

 

Following exposure to test item, the mean cell viability was 79.0% compared to the negative control (after adjustment for non-specific MTT reduction). 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.

 

CONCLUSION

Results from the in vitro EPISKIN (SM) indicate that the test item is non-corrosive to skin.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Irritation / corrosion parameter:
% tissue viability
Value:
79
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 October 2016 to 07 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiSkin SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Source strain:
other: adult
Justification for test system used:
INTRODUCTION
- 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)
- The test is designed to predict and classify the skin irritant potential of chemicals, formulations, products and mixtures according to chemical safety regulations, using the reconstructed human epidermis model EPISKIN (SM) and parameters related to skin irritation.
- EPISKIN (SM) is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller C. et al., 2002, Mosmann T., 1983). The reduction of cell viability in treated tissues is compared to negative controls and expressed as a %. The % reduction in viability is used to predict the irritation potential of the test item.
- Note that the OECD 439 is specifically a test for dermal irritation, it is not a test for corrosivity. A negative or positive result in this assay does not necessarily mean that the test item is non-corrosive.
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
- Human skin: EPISKIN (SM) (Manufacturer: SkinEthic, France, Batch No.: 16-EKIN-040, Expiry Date: 10 October 2016) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
- Quality control: EPISKIN (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkin (SM) Test Kits used in the present study) and are documented in Appendix 2 (attached).
- The EPISKIN (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

KIT CONTENTS
- Units: EPISKIN (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
- Plate: 12-well assay plate
- Punch: EPISKIN (SM) biopsy punch for easy sampling of epidermis.
- Medium: Flask of sterile “Maintenance Medium” (Batch No.: 16 MAIN3 067; Exp. Date: 12 October 2016); Flask of sterile “Assay Medium” (Batch No.: 16 ESSC 043; Exp. Date: 12 October 2016).

NUMBER OF REPLICATE WELLS
- In this assay, three replicates were used for the test item.
- Three negative controls and three positive controls were also run in each assay.
- Furthermore, as the test item was coloured, two additional test item-treated tissues were used for the non-specific OD (optical density or absorbance) evaluation.

KIT RECEPTION
- The pH of the agar medium used for transport was checked by checking the colour of the medium (orange colour = good; yellow or violet colour = not acceptable)
- The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40 °C (the colour change is irreversible, independent of the length of the period above 40 °C; white colour = good; grey or black colour = not acceptable)
- The kits were found to be in good order at reception.

STORAGE
- The EPISKIN (SM) kit was kept in its packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2 to 8 °C until the initiation of the test.

ADDITIONAL MATERIALS
- Details of chemicals used in the performed experiments are summarised in the table attached.
- MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 04 October 2016) was stored in refrigerator (2 to 8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.
- Acidified isopropanol: Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

INDICATOR FOR POTENTIAL FALSE VIABILITY
- Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.
- If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement.
- Approximately 10 μL of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded (test items which do not react with MTT: yellow; test items reacting with MTT: blue or purple).
- After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.
- Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol (simulating a tissue humid environment; water is the environment during exposure, isopropanol is the extracting solution). As the test items had an intrinsic colour, further evaluation to detect colouring potential was necessary. Non-specific Colour % (NSC living %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues.
- Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non specific OD evaluation. This tissue followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

PRE-INCUBATION (Day -1)
- 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.

APPLICATION AND RINSING (Day 0) – TEST ITEM
- Approximately 10 μL of test item were 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.

APPLICATION AND RINSING (Day 0) – NEGATIVE AND POSITIVE CONTROLS
- A 50 μL volume 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).
- The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.3 to 24.8 °C).
- After the 15 minutes incubation time, the EPISKIN (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 (Day 2)
- After the 42 hour incubation, all EPISKIN (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).
- The transferred EPISKIN (SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere, protected from light.

FORMAZAN EXTRACTION (Day 2)
- 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 (approximately 150 rpm) for formazan extraction.

CELL VIABILITY MEASUREMENTS (Day 2)
- Following the formazan extraction, 2 × 200 μL samples 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.
- The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, 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.

CALCULATION OF VIABILITY PERCENTAGES

BLANK
- The mean of the 6 blank OD values was calculated.

NEGATIVE CONTROL
- Individual negative control OD values (NC raw) were corrected with the mean blank OD using the equation OD Negative Control (OD NC) = OD NC raw – OD blank mean.
- The corrected mean OD of the 3 negative control values was also calculated: this corresponds to 100% viability.

POSITIVE CONTROL
- Individual positive control OD values (PC raw) were corrected with the mean blank OD using the equation OD Positive Control (OD PC) = OD PC raw – OD blank mean.
- The mean corrected OD of the 3 positive control values was calculated.
- The % viability for each positive control replicate was calculated relative to the mean negative control using the equations Positive Control 1 % = (OD PC1 / Mean OD NC) x 100, Positive Control 2 % = (OD PC2 / Mean OD NC) x 100 and Positive Control 3 % = (OD PC3 / Mean OD NC) x 100.
- The mean value of the 3 individual viability % for positive control was calculated using the equation Mean PC % = (%PC1 + %PC2 + %PC3) / 3.

TEST ITEM
- Individual test item OD values (TT raw) were corrected with the mean blank OD using the equation OD Treated Tissue (OD TT) = OD TT raw – OD blank mean.
- The corrected mean OD of the 3 test item values was calculated.
- The % viability for each test item replicate was calculated relative to the mean negative control using the equations Treated Tissue 1 % = (OD TT1 / Mean OD NC) x 100, Treated Tissue 2 % = (OD TT2 / Mean OD NC) x 100 and Treated Tissue 3 % = (OD TT3 / Mean OD NC) x 100.
- The mean value of the 3 individual viability % for test item was calculated using the equation Mean TT % = (%TT1 + %TT2 + %TT3) / 3.

DATA CALCULATIONS FOR TEST ITEMS HAVING MTT-INTERACTING POTENTIAL
- Test items that interfere with MTT can produce non-specific reduction of the MTT. In this case, additional control samples were used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value was corrected by the result of the additional controls before calculation of viability% using the equation Non specific MTT reduction calculation (NSMTT%) = [(OD KT – OD KNC) / OD NC] x 100 where OD KNC = negative control treated killed tissues OD; OD KT = test item treated killed tissues OD; OD NC = negative control OD.
- If NSMTT% is ≤ 30%, then true MTT metabolic conversion (TOD TT) had to be undertaken using the equation TOD TT = [OD TT – (OD KT – OD KNC)] where OD TT = test item treated viable tissues.
- The % relative viability (%RV) for each test item replicate was calculated relative to the mean negative control using the equations %RV1 = [TOD TT1 / Mean OD NC] x 100, %RV2 = [TOD TT2 / Mean OD NC] x 100 and %RV3 = [TOD TT3 / Mean OD NC] x 100.
- The mean value of the 3 individual relative viability % results for test item was calculated using the equation Mean Relative Viability % = (%RV1 + %RV2 + %RV3) / 3.
- If NSMTT% is > 30% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

DATA CALCULATION FOR TEST ITEMS HAVING COLOURING POTENTIAL
- For test items detected as able to stain the tissues, the non-specific OD due to the residual chemical colour (unrelated to mitochondrial activity) is evaluated and subtracted before calculation of the “true” viability % using the equation Non Specific Colour % (NSC living %) = (Mean OD CTV / Mean OD NC) x 100 where OD CTV = test item treated viable tissue (not incubated with MTT); OD NC = negative control OD (incubated with MTT).
- If NSC living % is ≤ 5 % then the normal calculation mode was used.
- If NSC living % is > 5 % and ≤ 50 %, then true MTT metabolic conversion (TOD TT) had to be undertaken using the equation TOD TT = [OD TV – OD CTV] where ODTT = test item treated viable tissue (incubated with MTT); OD CTV = test item treated viable tissue (not incubated with MTT).
- The % relative viability (%RV) for each test item replicate was calculated relative to the mean negative control using the equations %RV1 = [TOD TT1 / Mean OD NC] x 100, %RV2 = [TOD TT2 / Mean OD NC] x 100 and %RV3 = [TOD TT3 / Mean OD NC] x 100.
- The mean value of the 3 individual relative viability % results for test item was calculated using the equation Mean Relative Viability % = (%RV1 + %RV2 + %RV3) / 3.
- If NSMTT% is > 30% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

DATA CALCULATION FOR TEST ITEMS HAVING BOTH MTT-INTERACTING AND COLOURING POTENTIAL
- Test items detected as able to both stain the tissues and interfere with MTT also require a third set of controls before calculation of the “true” viability %.
- Non Specific Colour % with killed tissues (NSC killed %) = (Mean OD CTK / Mean OD NC) x 100 where OD CTK = test item treated killed tissues (not incubated with MTT); OD NC = negative control OD (incubated with MTT) and TOD TT = [OD TT – (OD KT – OD KNC) – Mean OD CTV + Mean OD CTK] where OD TT = test item treated viable tissues (incubated with MTT); OD KT = test item treated killed tissues OD; OD KNC = negative control killed tissues OD; OD CTV = test item treated viable tissues (not incubated with MTT); OD CTK = test item treated killed tissues (not incubated with MTT).
- The % relative viability (%RV) for each test item replicate was calculated relative to the mean negative control using the equations %RV1 = [TOD TT1 / Mean OD NC] x 100, %RV2 = [TOD TT2 / Mean OD NC] x 100 and %RV3 = [TOD TT3 / Mean OD NC] x 100.
- The mean value of the two individual relative viability % results for test item was calculated using the equation Mean Relative Viability % = (%RV1 + %RV2 + %RV3) / 3.

VALIDITY OF THE TEST
- The mean OD value of the three negative control tissues should be between 0.6 and 1.5, and the standard deviation value (SD) of the % viability values should be ≤ 18.
- The acceptable mean percentage viability range for positive controls is 0 to 40% and the
standard deviation value (SD) of the % viability values should be ≤ 18.
- The SD calculated from individual % tissue viability values of the three test item treated
replicates should be ≤ 18.
- The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.

INTERPRETATION OF TEST RESULTS
- The irritation potential of test items can be classified according to the United Nations globally Harmonized System of Classification and Labelling of Chemicals, and a
similar system is used in CLP.
- In the present study, the irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item.
- The test item is considered to be non-irritant to skin if the mean relative viability after 15 minutes exposure and 42 hours post exposure incubation is > 50 % of the negative control. Note: If there is clear evidence that the test item is not corrosive, then it can be determined as No Category according to the UN GHS. It is plausible that some weaker corrosives could be classified as non-irritant in this in vitro assay.
- The test item is considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post exposure incubation is ≤ 50 % of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL of test item
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Value:
6.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ADDITIONAL CONTROLS
- As no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, the test material did not interact with MTT and additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
- As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues were 0.025, Non-specific Colour % was calculated as 3.2 % (see Table 1, attached). This value was below 5 %, therefore additional data calculation was not necessary.

VIABILITY RESULTS
- The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 2 (attached).
- The mean OD value for the test item treated skin samples showed 6.6 % relative viability.

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 (0.764). Standard deviation of the viability results for negative control samples was 6.8.
- The positive control treated tissues 5.3 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.2.
- The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 2.0.
- The mean OD value of the blank samples (acidified isopropanol) was 0.045.
- All these parameters met the acceptability criteria, therefore the study was considered to be valid.
- Historical control data are presented in Appendix 3 (attached).
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Mean cell viability was 6.6 % compared to the negative control and the test item is considered to be a skin irritant.
Executive summary:

GUIDELINE

An in vitro skin irritation test of test item was performed in a reconstructed human epidermis model. EPISKIN (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 irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

 

METHODS

Disks of EPISKIN (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 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 (NSC living) from the test item. For each treated tissue, the viability was expressed as a % relative compared to the negative control. If the mean relative viability after 15 minutesexposure and 42 hours post incubation is less or equal (≤) to 50% of the negativecontrol, the test item is considered to be irritant to skin.

 

RESULTS

 

Following exposure to test item, the mean cell viability was 6.6 % compared to the negative control. This is below the threshold of 50%, therefore the test item was considered as being irritant to skin (removal of test item at 15 minutes was not fully achieved due to the physical characteristics of the test item). The experiment met the validity criteria, therefore the study was considered to be valid.

 

CONCLUSION

Mean cell viability was 6.6 % compared to the negative control and the test item is considered to be a skin irritant.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Irritation / corrosion parameter:
% tissue viability
Value:
6.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 February 2017 to 3 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
various (see below)
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
various (see below)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Deviations:
yes
Remarks:
various (see below)
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Type of coverage:
occlusive
Preparation of test site:
clipped
Remarks:
followed by shaving
Vehicle:
unchanged (no vehicle)
Controls:
other: The untreated skin of each animal served as a control.
Amount / concentration applied:
0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
14 days
Number of animals:
Three
Details on study design:
MATERIALS USED FOR TREATMENT

INZULIN E.H. FECSKENDŐ (Syringe, 1mL)
Batch number: 150502
Expiry date: May 2020
Supplier: WOLF Orvosi műszer (Hungary) KFT

STERIL MULL-LAP (sterile gauze pads)
Batch number: 1510675
Expiry date: November 2020
Produced by: Dispomedicor Zrt, Hungary

Betasilk (adhesive tape)
Batch number: 9257
Expiry date: March 2021
Produced by: GS Medical Ltd., Dublin

Ramofix (flexible bandage)
Batch number: 2016 JAN 11
Expiry Date: 29 January 2021
Produced by: Ramofix Trade Kft, Hungary.

Unguentum hydrophilicum nonionicum Ph.Hg. (washbasin ointment)
Batch number: 18200 616
Expiry date: June 2020
Produced by: Naturland Magyarország Kft.

MATERIALS USED FOR EUTHANASIA

Ketanest 100 mg/ml (Ketamine)
Batch No.: H1023-10
Expiry date: March 2019
Supplier: bela-pharm Gmbh & Co. KG

Nerfasin 20 mg/ml, (Xylazin)
Batch number: 16A134
Expiry date: December 2018
Supplier: Le Vet B.V

Release, 300 mg/ml pentobarbital sodium
Batch number: 106075
Expiry date: July 2018
Produced by: Alpha-Vet Állatgyógyászati Kft.

EXPERIMENTAL ANIMALS
- Species and strain: New Zealand White rabbits
- Source: S&K-LAP Kft. 2173 Kartal, Császár út 135, HUNGARY
- Justification of strain: The New Zealand White albino rabbit is one of the standard strains used for acute irritation toxicity studies.
- Animal health: Only animals in acceptable health condition were used for the test.
- Number of animals: 3
- Sex: Male
- Age of animals at treatment: 11 to 13 weeks old
- Body weight range at the beginning of the in-life phase: 3289 – 3635 g
- Body weight range at the end of the in-life phase: 3435 – 3791 g
- Date of receipt: 18 January 2017
- Acclimatisation time: 20 or 27 days
- Animal identification: The animals were identified by engraved ear tags. The cages were marked with individual identity cards with information about study code, sex, cage number, dose and individual animal number.

HUSBANDRY
- Number of animal room: 033
- Housing/Enrichment: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.
- Lightning period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- Temperature: 20.1 – 24.3 °C
- Relative humidity: 36 – 79 %
- Ventilation: 15-20 air exchanges/hour
- Temperature and relative humidity values were measured continuously. The measured range was checked at least daily during the acclimatisation and experimental phases.

FOOD AND FEEDING
- Animals received UNI diet for rabbits produced by Cargill Takarmány Zrt., H-5300 Karcag, Madarasi út, Hungary, ad libitum. Animals were provided with the following batches:
(i) Batch number: 0003801145, expiry date: 01 March 2017
(ii) Batch number: 0003925332, expiry date: 23 April 2017
- Details of the diet used were archived with the raw data but not reported.

WATER SUPPLY AND WATER QUALITY
- The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system.
- The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A. u. 36., Hungary). Copies of the relevant Certificates of Analysis are retained in the archives at CiToxLAB Hungary Ltd.

DOSAGE
- A volume of 0.5 mL of the test item/application site was applied to the skin of the experimental animals.
- The test items were applied as a single dose.
- The untreated skin of each animal served as a control.

APPLICATION OF TEST ITEM
- Patch testing was used to detect primary irritating effects of the test item. Three male animals in acceptable health condition were selected for the test.
- Approximately 24 hours prior to the test, the hair was clipped from the back of the animals. Removal of hair was performed in two steps. The majority of hair was clipped with an electronic hair clipper and the remaining hair was moistened with water and shaved with a razor.
- For ethical reasons, an initial test was performed using a single animal with additional checks.
- The first patch was removed after three minutes. As no serious skin reaction was observed, a second patch was applied at a different site and removed after one hour. The observations at this stage indicated that exposure was allowed to extend to four hours, a third patch was applied and removed after four hours, and the response was graded.
- There was no corrosive effect, no significant systemic toxicity and no other severe local effects per application site observed, then two additional animals were included in this study.
- The test item was applied to an area of approximately 6 cm2 of intact skin.
- Sterile gauze pads were placed on the skin of rabbits. These gauze pads were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The entire trunk of the animals was wrapped with plastic wrap held in place with an elastic stocking for 4 hours.
- The degree of irritation was observed and scored at specified intervals by
examination of the treated area (each application site). The untreated skin of each animal served as control skin. The duration of the study was sufficient to evaluate the reversibility or irreversibility of the effects. At termination, animals were sacrificed.

DURATION OF EXPOSURE
- Duration of exposure: 3 minutes, 1 hour or four hours.
- After the application, the remaining test item was removed by gently washing with Unguentum hydrophilicum nonionicum Ph. Hg. (washbasin ointment) and body temperature
water.

CLINICAL OBSERVATIONS
- Animals were examined for signs of erythema and oedema, and the responses scored at 3 and/or 60 minutes and then at 24, 48, 72 hours, 1 and 2 weeks a after patch removal.

SCORING AND ASSESSMENT OF LOCAL REACTIONS
- The dermal irritation scores were evaluated according to the scoring system by Draize (1959) shown in Appendix 1 (attached).

MEASUREMENT OF BODY WEIGHT
- Body weights were recorded at the beginning and at the end of experiment.

TERMINATION
- At the end of the observation period, euthanasia of the animal was by intramuscular injections of Ketamin 10 % and Xylazin 2 % followed by i.v. Pentobarbital sodium anaesthesia.
- Death was verified by checking pupil and cornea reflex, absence of respiration and pulse.
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
mean score
Time point:
24/48/72 h
Score:
0.67
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2748 (male)
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
7 d
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2748 (male)
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
14 d
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2748 (male)
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
mean score
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2712 (male)
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
7 d
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2712 (male)
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
14 d
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2712 (male)
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
mean score
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2719 (male)
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
7 d
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2719 (male)
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
14 d
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2719 (male)
Irritation parameter:
edema score
Basis:
other: all animals
Remarks:
mean score
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: animals 2748, 2712 and 2719 (male)
Irritation parameter:
edema score
Basis:
other: all animals
Time point:
14 d
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: animals 2748, 2712 and 2719 (male)
Irritant / corrosive response data:
RESULTS
- No mortality observed during the study.
- Scoring of erythema and oedema formation in the initial test is shown in Table 1 (attached).
- Scoring of erythema and oedema formation after 4 hours exposure is shown in Table 2 (attached).
- Mean skin irritation scores (24, 48 and 72 hours) are shown in Table 3 (attached).
- There was no test item related effect on body weight (see Table 4, attached).
- No test item related clinical signs were noted during general daily examinations.
- Initial test (animal 2748): The first patch was removed after three minutes. As no serious skin reaction was observed (scores were zero), a second patch is applied at a different site and
removed after one hour. As the observations at this stage indicated (scores were zero) that the exposure was extended to four hours, a third patch was applied and removed after four hours, and the response was graded. At observation 1 and 24 hours after patch removal, there were no observed clinical signs noted on the test site of the treated animal. At 48, 72 hours, 1 and 2 weeks observation after patch removal, very slight erythema (score 1) was observed.
- Second test animal (2712): At observation 1 hour after patch removal, there were no observed clinical signs noted on the test site of the treated animal. At 24, 48, 72 hours, 1 and 2 weeks observation after patch removal, very slight erythema (score 1) was observed. At 1 week observation time point, dry and cracked skin was also noted.
- Third test animal (2719): At observation 1 hour after patch removal, there were no observed clinical signs noted on the test site of the treated animal. At 24, 48, 72 hours, 1 and 2 weeks observation after patch removal, very slight erythema (score 1) was observed. At 1 week observation time point, dry and cracked skin was also noted.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Based on the scheme devised by Draize (1959), the test item was considered to be a mild irritant.
Executive summary:

GUIDELINE

An acute skin irritation study was performed on New Zealand White rabbits in accordance with OECD Guidelines for Testing of Chemicals 404 (28th July 2015), Commission Regulation (EC) No 440/2008, B.4 (L 142, 30 May 2008) and OPPTS 870.2500 (EPA 712-C-98-196) August 1998.

 

METHODS

A volume of 0.5 mL of the test item was applied to the skin of the experimental animals. The test items were applied as a single dose. Sterile gauze pads were placed on the skin of rabbits. These gauze pads were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The trunk was wrapped in clear plastic with medical tubing used to hold the patch in place. The untreated skin of each animal served as control. After the application, the remaining test item was removed by gently washing with Unguentum hydrophilicum nonionicum Ph. Hg. (washbasin ointment) and body temperature water. The treated skin surfaces were examined at 3 minutes and/or 1, 24, 48, 72 hours, 1 and 2 weeks after the patch removal. Additional general examinations were performed daily.

 

RESULTS

There was no mortality during the observation period and no test item-related clinical signs or effects on body weight. For animal 1, at 48, 72 hours, 1 and 2 weeks after patch removal, very slight erythema (score 1) was observed. For animal 2, at 24, 48, 72 hours, 1 and 2 weeks after patch removal, very slight erythema (score 1) was observed. At the 1-week observation, dry and cracked skin was also noted. For animal 3, at 24, 48, 72 hours, 1 and 2 weeks after patch removal, very slight erythema (score 1) was observed. At the 1-week observation, dry and cracked skin was also noted. The observed erythema did not reverse in any of the three animals during the 14-day observation period.

 

CONCLUSION

Based on the scheme devised by Draize (1959), the test item was considered to be a mild irritant.

Endpoint:
skin irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
mean score
Time point:
24/48/72 h
Score:
0.67
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2748 (male)
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
7 d
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2748 (male)
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
14 d
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2748 (male)
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
mean score
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2712 (male)
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
7 d
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2712 (male)
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
14 d
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2712 (male)
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
mean score
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2719 (male)
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
7 d
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2719 (male)
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
14 d
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 2719 (male)
Irritation parameter:
edema score
Basis:
other: all animals
Remarks:
mean score
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: animals 2748, 2712 and 2719 (male)
Irritation parameter:
edema score
Basis:
other: all animals
Time point:
14 d
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: animals 2748, 2712 and 2719 (male)
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May 2016 to 22 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
chicken
Strain:
other: COBB 500 in Experiment I and ROSS 308 in Experiment II
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Approximately 30 μL of test item
Duration of treatment / exposure:
10 seconds
Number of animals or in vitro replicates:
Three each in experiments I and II
Details on study design:
INTRODUCTION
- The Enucleated Eye Test with isolated eyes of chickens is a well validated and accepted in vitro test system. It has been recognised as a valuable alternative to the Draize eye irritation test, because it represents a test system nearest to the in vivo test, without the need to use live animals. It can also be used as a screening tool for corrosivity/severe irritancy to avoid unacceptable effects in vivo. In the Isolated Chicken Eye Test (ICET) the test compound is applied in one single dose onto the cornea of isolated eyes. Chicken heads are obtained from a veterinary-inspected, commercial slaughter-house, processing chickens for human consumption.
- This method can provide detailed information about the effects of test items on the cornea, and can be used to identify chemicals not requiring classification for eye irritation, or for serious eye damage, as defined by the UN GHS (UN GHS nonclassified or UN GHS Category 1). The test is described in OECD No. 438 and is approved by international regulatory agencies as a replacement for the identification of non-irritant, corrosives/severe irritants in the in vivo Rabbit Eye Assay (OECD No. 405).
- The procedure was first performed on 3 test item treated eyes, 3 positive control eyes and 1 negative control eye (Experiment I.) then on a further 3 test item treated eyes, 3 positive control eyes, 1 negative control eye and 1 negative control eye with more excessive rinsing and extension observation time (Experiment II.) due to uncertainty for part of the results in the first run.

TEST ITEM SOLUBILITY AND FORMULATION
- Solubility of the test item in physiological saline was tested prior to the Experiment I (30 μL test item in 1 mL saline). The test item did not dissolve in physiological saline.
- The test item was applied as supplied, no formulation was required.

POSITIVE CONTROL
- The material was diluted with distilled water (Supplier: Teva Co., Lot number: 6820914, Expiry date: September 2017 in Experiment I and Lot number: 7170914, Expiry date: September 2017 in Experiment II) to achieve a final concentration of 5% (w/v).

SUBSIDIARY MATERIAL
- Experiment I: Fluorescein sodium (Merck; lot number K46664387; expiry date 31 May 2020). This material was mixed with physiological saline (Manufacturer: B. Braun Melsungen AG, Lot number: 52532Y05-2, Expiry date: 31 May 2018) to achieve a final concentration of 2% (w/v). The resulted solution was stored at room temperature (Dispensary code: S43100, Expiry date: 02 June 2016.).
- Experiment II: Fluorescein sodium 10 % w/v (ALCON; lot number 250817F; expiry date 31 May 2017). This fluorescein solution was mixed with physiological saline (Manufacturer: B. Braun Pharmaceuticals SA, Batch number: 52532Y05-2; Expiry date: 31 May 2018) to achieve a final concentration of 2% (w/v). The resulted solution was stored at room temperature (Dispensary code: S43102, Expiry date 07 July 2016).

CHICKEN HEADS COLLECTION AND TRANSPORT
- Source: TARAVIS KFT, 9600 Sárvár, Rábasömjéni út.129., Hungary.
- Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
- After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.

SELECTION OF EYES
- After removing the head from the plastic box, it was put on soft paper.
- The eyelids were carefully cut away with scissors, avoiding damaging the cornea.
- One small drop of 2 % (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. - If the cornea was in good condition, the eyeball was carefully removed from the orbit.

PREPARATION OF EYES
- The eye ball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity.
- Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EYE EXAMINATION AND ACCLIMATISATION TIME
- The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head) avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
- The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining ( > 0.5) or corneal opacity score (> 0.5) were rejected. The cornea thickness was measured and any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, the acclimatisation started and was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5°C) during the acclimatisation and treatment periods.

IDENTIFICATION
- The eyes were identified by chamber number, marked on the door of the chamber.

BASELINE ASSESSMENTS
- At the end of the acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye.
- The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time.
- No changes in thickness (0.0%) were observed in the eyes.
- Following the equilibration period, the fluorescein retention was measured.
- Baseline values were required to evaluate any potential test item related effect after treatment.
- All eyes were considered to be suitable for the assay.

TREATMENT
- After the zero reference measurements, the eye (in its retainer) was removed from the chamber, held in a horizontal position and the test item was applied onto the centre of the cornea, taking care not to damage or touch the cornea.
- Approximately 30 μL of test item was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance.
- Negative control eye and negative control eye with more excessive rinsing were treated with 30 μL of physiological saline; positive control eyes were treated with 30 μL 5% (w/v) Benzalkonium chloride solution.

TEST ITEM REMOVAL
- The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test item if possible.
- Additional gentle rinsing with 20 mL saline was performed at each time point when remaining test item or control material was observed on the cornea.
- Note: More excessive rinsing with 31.7 °C physiological saline was applied in Experiment II.

OBSERVATION AND ASSESSMENT OF CORNEAL EFFECTS
- The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ± 5 minutes were considered acceptable.
- Corneal thickness and corneal opacity were measured at all time points.
- Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse, using a Haag-Streit BP 900 slit-lamp microscope.

EVALUATION
- Corneal swelling was calculated using the formulae CS at time t = (CT at time t – CT at t=0 / CT at t = ) x 100 and Mean CS at time t = (FECS at time t + SECS at time t + TECS at time t) / 3 where CS = cornea swelling; CT = cornea thickness; FECS at time t = first eye cornea swelling at a given time point; SECS at time t = second eye cornea swelling at a given time-point; TECS at time t = third eye cornea swelling at a given time-point.
- Small negative numbers for swelling (0 to -5%) following application are evaluated as class I. Large negative numbers (> 12% below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5% to -12% are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).
- Cornea opacity was calculated according to the formulae ACO at time t = CO at time t – CO at t=0 and Mean ΔCO max = (FECO max 30 min to 240 min + SECO max 30 min to 240 min + TECO max 30 min to 240 min) / 3 where CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post treatment rinse; CO at t=0 = baseline cornea opacity; ΔCO at time t = difference between cornea opacity at t time and cornea opacity baseline; FECO = first eye cornea opacity; SECO = second eye cornea opacity; TECO= third eye cornea opacity; max 30min to 240min = maximum opacity of the individual eye at 30 to 240 minutes minus
baseline cornea opacity of the individual eye.
- Fluorescein retention was calculated according to the formulae ΔFR at time t = FR at time t – FR at t=0 and Mean ΔFR = (FEFR 30 min + SEFR 30 min + TEFR 30 min) / 3 where FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse; FR at t=0 = baseline fluorescein retention; ΔFR at time t = difference between fluorescein retention at t time and fluorescein retention baseline; FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention; SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention; TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention.

RETENTION OF CHICKEN EYES
- At the end of the procedure, the corneas from the eyes were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10% neutral buffered formalin, Manufacturer: Reanal, Batch number: KTM14051, Expiry date: October 2017 in Experiment I. and Batch number: KTM15011, Expiry date: February 2018 in Experiment II.) was used for potential histopathology and stored at room temperature.
Irritation parameter:
other: overall ICE class
Run / experiment:
Experiment I
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 1 x III, 1 x IV
Remarks:
test item
Irritation parameter:
other: overall ICE class
Run / experiment:
Experiment II
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 2 x III, 1 x IV
Remarks:
test item
Other effects / acceptance of results:
TEST ITEM
- Individual data for Experiments I and II are shown in Tables 1.1 and 1.2 (see Appendix 1, attached).
- The mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change are attached for Experiments I and II. The conclusion on eye irritancy was based on the OECD guideline quantitative assessments, shown in Appendix 2 (attached).
- Details of data interpretation for Isolated Chicken Eye (ICE) Class are given in Appendix 2 and 3 (attached). The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention ICE classes are used for EC and GHS classification.
- In Experiment I, test item was stuck on all cornea surfaces (confluent layer) during the full post treatment period and could not be rinsed off and all three cornea surfaces were not cleared at 240 minutes after the post-treatment rinse, and as a result, the corneal opacity, the corneal swelling and the fluorescein retention could not be properly evaluated.
- Due to the equivocal results, an additional experiment was performed for clarification as agreed by the sponsor. The experimental conditions were the same as in the first test, except for more excessive rinsing with physiological saline (31.7 °C) and an extension of the observation time. Moreover an additional control (standard controls and one negative control with similar excessive rinsing) demonstrated that the additional washing had no adverse effect.
- In Experiment II, all three cornea surfaces were not cleared fully at 300 minutes after the post-treatment rinse, however the corneal thickness, corneal opacity and fluorescein retention was assessed on the clear areas of eyes. The areas of test item stuck to the cornea could mask corneal effects underneath the residues and could also potentially result in mechanical damage in vivo. Based on this the scores situated the test item as less than, but close to, the cut-off for a severe irritant. The test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that an in vivo study is required for proper classification.
- A summary table showing UN GHS classification information relating to the test item is attached.

POSITIVE CONTROL
- Individual data for Experiments I and II are shown in Tables 1.3 and 1.4 (see Appendix 1, attached).
- The positive controls 5% (w/v) Benzalkonium chloride solutions were classified as severely irritating, UN GHS Classification: Category 1 (see results for Experiments I and II, attached).

NEGATIVE CONTROL
- Individual data for Experiments I and II are shown in Tables 1.5, 1.6 and 1.7 (see Appendix 1, attached).
- The negative controls Physiological saline solutions were classified as non-irritating, UN GHS Classification: Non-classified (see results for Experiments 1 and II, attached).

VALIDITY OF THE TEST
- Historical control data is attached.
- The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historic data. This experiment was considered to be valid.
- For the classification, the guideline clearly says that alcohol containing test items may be over-predicted. A more reliable result may be obtained following histopathology evaluation of the corneas.
Interpretation of results:
study cannot be used for classification
Conclusions:
The test item was not classified as a severe irritant and not classified as non-irritant using the in vitro isolated chicken eye test method. It was therefore concluded that an in vivo study was required.
Executive summary:

GUIDELINE

An in vitro eye irritation study of the test item was performed in isolated chicken eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 (26 July 2013).

 

METHODS

After the zero reference measurements, the eye was held in horizontal position and approximately 30 μL of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 μL Benzalkonium chloride 5% solution. The negative control eye was treated with 30 μL of physiological saline (9% (w/v) NaCl solution). Examinations in Experiment I were three test item treated eyes, three positive control treated eyes and one negative control treated eye. In Experiment II, three test item treated eyes with more excessive rinsing, three positive control treated eyes, one negative control treated eye and one negative control treated eye with more excessive rinsing were examined. The observation time was also extended in Experiment II.

 

RESULTS

Results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

 

Test item was stuck on all cornea surfaces (confluent layer) during the full post treatment period and could not be rinsed off (all three cornea surfaces were not cleared at 240 minutes after the post-treatment rinse) and, as a result, the corneal opacity, the corneal swelling and the fluorescein retention could not be properly evaluated.

 

Due to the equivocal results, an additional experiment was performed for clarification as agreed by the sponsor.The experimental conditions were the same as in the first test, except for more excessive rinsing with physiological saline (31.7 °C) and an extension of the observation time. Moreover an additional control (standard controls and one negative control with similar excessive rinsing) demonstrated that the additional washing had no adverse effect.

 

Experiment I: Severe corneal swelling was observed during the four-hour observation period on test item treated eyes. Cornea opacity change could not be evaluated. Moderate fluorescein retention change (severity 2) was noted on three eyes. Test item was stuck on all cornea surfaces (confluent layer) after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

 

Experiment II: Moderate corneal swelling was observed during the five-hour observation period on test item treated eyes. Moderate cornea opacity change (severity 1 or 2) was observed on three eyes. Severe fluorescein retention change (severity 2 or 3) was noted on three eyes. These scores situate the test item as below, but close to, the cut-off for a severe irritant. Relatively large areas of the test item could be rinsed off, but small areas of the test item had material still stuck on all cornea surfaces even with after the post- excessive rinsing. All three cornea surfaces were not cleared fully at 300 minutes after the post-treatment rinse. The areas of test item stuck to the cornea could mask corneal effects underneath the particles and could also potentially result in mechanical damage in vivo.

 

CONCLUSION

The test item was not classified as a severe irritant and not classified as non-irritant using the in vitro isolated chicken eye test method. It was therefore concluded that an in vivo study was required.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Irritation parameter:
other: overall ICE class
Run / experiment:
Experiment I
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 1 x III, 1 x IV
Remarks:
test item
Irritation parameter:
other: overall ICE class
Run / experiment:
Experiment II
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 2 x III, 1 x IV
Remarks:
test item
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 September 2016 to 16 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
maximum temperature exceeded with no impact on outcome of study or interpretation of results (see below)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
yes
Remarks:
maximum temperature exceeded with no impact on outcome of study or interpretation of results (see below)
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
EXPERIMENTAL ANIMAL
- Species and strain: New Zealand White rabbit
- Source: S&K-LAP Kft, 2173 Kartal, Császár út 135, Hungary.
- Justification of strain: The New Zealand White rabbit is one of the standard strains used for acute irritation toxicity studies.
- Animal health: The animal used for the test was in acceptable health condition. Both eyes of the animal provisionally selected for testing were examined prior to starting the study. Animals showing eye irritation, ocular defects or pre-existing corneal injury were not used.
- Number of animals: 1 animal
- Age of animal at treatment: 15 weeks old
- Sex: naive male
- Body weight: on the day of treatment: 4012 g; before euthanasia on Day 14: 4370 g
- Date of receipt: 20 July 2016
- Acclimatisation time: at least 43 days
- Animal identification: The individual identification was by engraved ear tag. The cage was marked with individual identity card with information about study code, sex, dose, cage number and individual animal number.

HUSBANDRY
- Animal health: Only healthy animals were used for the test. The health status was certified by the staff veterinarian.
- Number of animal room: 032
- Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- Temperature during the study: 21.64 – 23.90 °C
- Relative humidity during the study: 41.87 – 64.12 %
- Housing/Enrichment: Rabbit was individually housed in AAALAC approved metal wire rabbit cage. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.
- Ventilation: 15-20 air exchanges/hour
- The temperature and relative humidity values were measured continuously. The measured range was checked at least daily during the acclimatisation and experimental phases.

FOOD AND FEEDING
- Animals received UNI diet for rabbits produced by Cargill Takarmány Zrt., H-5300 Karcag, Madarasi út 0399, Hungary, ad libitum (batch 0003304513, expiry date: 04 August 2016 and 0003403537, expiry date: 16 September 2016).
- The details of the diet used were archived with the raw data but not reported.

WATER SUPPLY AND QUALITY CONTROL OF WATER
- The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system. The quality control analysis is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u. 36, Hungary). The quality control results are retained in the archives of CiToxLAB Hungary Ltd.
- The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated right eye served as control
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
Single dose with treated eye rinsed with physiological saline solution one hour and 24 hours after the application of test item due to ocular effects observed.
Observation period (in vivo):
2 weeks
Number of animals or in vitro replicates:
One
Details on study design:
IDENTIFICATION OF pH
- The pH value of the test item was assessed by measurement of the supernatant from a 1% w/v mixture in distilled water according to CIPAC MT75. The pH was found to be 4.5 for the test item, so the test item is permitted for use in animal studies.

IN VITRO STUDY RESULTS
- An in vitro eye irritation assay in the isolated chicken eyes concluded that the test item is not classified as a severe irritant and not classified as non-irritant. An in vivo study was therefore required for purposes of classification.

PRE-STUDY EXAMINATION
- One male animal in acceptable health condition was selected for the test. Care was taken to select only those animals that had a normal eye condition and any with ocular lesions were rejected.

CHRONOLOGY OF ANIMAL USE
- Only one rabbit was treated with test item.
- Due to the local effects, the animal was euthanised for reasons of animal welfare and the study was terminated 2 weeks after treatment.
- Assessment of irreversibility was possible for 2 weeks and, after discussion with the veterinarian, it was clear that the process of eye damage was irreversible.

ANALGESIC AND ANAESTHETIC TREATMENT
- Sixty minutes (60 ±10 min) prior to test substance application, a systemic opiate analgesic was administered by subcutaneous injection (SC) under direct veterinary supervision.
- Five minutes (5 ±1.5 min) prior to test substance application, a topical ocular anaesthetic was applied to each eye (including the control eye to ensure direct comparison of any ocular observations).
- Eight hours (8 to 9 hr) after test substance application, a systemic opiate analgesic and a nonsteroidal anti-inflammatory drug (NSAID) were administered by SC injection under direct veterinary supervision. The systemic opiate analgesic was again injected approximately12 hours after the post-treatment analgesic and then every 12 hours, with NSAID injected approximately every 24 hours.
- Systemic opiate analgesic: Buprenorphine 0.01 mg/kg.
- Topical ocular anaesthetic: Humacain (oxybuprocaine) one-two drops per eye.
- Nonsteroidal anti-inflammatory drug: Loxicom 0.5 mg/kg.

ADMINISTRATION OF TEST ITEM
- Dosage: A single volume of the 0.1 mL of test item was administered to the left eye of the experimental animal.
- Application of test item: The test substance was placed in the conjunctival sac of the left eye of the animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for at least one second in order to prevent loss of the material. The untreated contralateral eye served as the control.
- Duration of exposure: Due to irritation scores of more than 2, the treated eye of the animal was rinsed with physiological saline solution one hour and 24 hours after the application of test item.

OBSERVATIONS AND SCORING
- The eyes were examined at 1, 24, 48 and 72 hours, 1 and 2 weeks after treatment and duration of the observation period was sufficient to identify reversibility or irreversibility of changes.
- The rabbit was examined for distress at least twice daily, with observations at least 6 hours apart. Clinical observations or signs of ill-health were recorded.
- Any clinical signs of toxicity or signs of ill-health during the study were recorded.
- At the end of the observation period, the animal was sacrificed by intramuscular injections of Ketamin 10% (Ketamidor) and Xylazin 2% (Primazin 2%) followed by i.v. pentobarbital sodium. Death was verified by checking pupil and corneal reflex and the absence of respiration.

SCORING AND ASSESSMENT OF LOCAL REACTION
- The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (02 October 2012) shown in Appendix 1 (attached).

MEASUREMENT OF BODY WEIGHT
- Individual body weight was recorded on the day of treatment and at the end of observation period of the experimental animal (see Table 2, attached).
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
mean score
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 4397 (male)
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
mean score
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Remarks on result:
other: animal 4397 (male)
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Remarks:
mean score
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not fully reversible within: 2 weeks
Remarks on result:
other: animal 4397 (male)
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
mean score
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
not fully reversible within: 2 weeks
Remarks on result:
other: animal 4397 (male)
Irritant / corrosive response data:
EXAMINATION OF EYE IRRITANCY
- No Initial Pain Reaction (IPR) was observed in this animal that was exposed to the test item. At subsequent time points no pain reaction (PR) was observed in this animal.
- At one hour after the application, conjunctival redness (score 2), chemosis (score 2), conjunctival discharge (score 3) and corneal opacity (score 2, area 4) were noted in the rabbit. Test item remained in the eye sac.
- At 24 hours after the application, conjunctival redness (score 3), chemosis (score 3), yellowish-white discharge (score 3), corneal opacity (score 2, area 4) and were noted in the rabbit.
- At 48 and 72 hours after the application, conjunctival redness (score 3), chemosis (score 3), yellowish-whitish and reddish discharge (score 3), corneal opacity (score 2, area 4) and were noted in the rabbit.
- At 1 week after the application, conjunctival redness (score 2), chemosis (score 2) and whitish discharge (score 3), corneal opacity (score 2, area 4) were noted in the rabbit.
- At 2 weeks after the application, conjunctival redness (score 1), chemosis (score 1), discharge (score 2) and corneal opacity (score 3, area 4) were noted in the rabbit. The cornea was more bulging than the control eye. Importantly, the pupil did not give any response for light and it was impossible to fully examine the iris at this time point. After discussion with the veterinarian, it was clear that the process of eye damage was irreversible.
- During the experiment, the control eye of the animal was symptom-free.
Other effects:
MORTALITY
- There was no mortality observed during the study.

BODY WEIGHT
- The body weight of the animal was considered to be within the normal range of variability. (see Table 2, attached).

GENERAL DAILY EXAMINATION
- There were no clinical signs observed that could be related to treatment.
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test item, applied to rabbit eye mucosa, caused significant conjunctival and corneal irritant effects. Due to the local effects, the animal was euthanised for reasons of animal welfare and the study was terminated 2 weeks after the treatment. No further test was deemed necessary.
Executive summary:

GUIDELINE

An acute eye irritation study was performed in the New Zealand White rabbit. The irritation effects of the test item were evaluated according to the Draize method (OECD No 405, 2012). One animal was used and the rabbit was treated with analgesic and anaesthetic as per the regulatory guideline.

 

METHODS

The test item was placed into the conjunctival sac of the left eye of the animal. A single amount of 0.1 mL of the test item was administered as a single dose and, as a result of ocular effects observed, the treated eye was rinsed with physiological saline at one hour and 24 hours after the application of test item. The untreated right eye served as control. The eyes were examined at 1, 24, 48, 72 hours, 1 and 2 weeks after application of the test item.

 

RESULTS

The general state and behaviour of the animal were normal throughout the study period. The body weights of the rabbit were considered to be within the normal range of variability.No Initial Pain Reaction (IPR)was observed in the animal that was exposed to the test item. At subsequent time points no pain reaction (PR) was observed in this animal.At one hour after the application,conjunctival redness (score 2), chemosis (score 2), conjunctival discharge (score 3) and corneal opacity (score 2, area 4) were noted in the rabbit. The test item remained in the eye sac.At 24 hours after the application,conjunctival redness (score 3), chemosis (score 3), yellowish-white discharge (score 3), corneal opacity (score 2, area 4) and were noted in the rabbit.At 48 and 72 hours after the application,conjunctival redness (score 3), chemosis (score 3), yellowish-whitish and reddish discharge (score 3), corneal opacity (score 2, area 4) and were noted in the rabbit.At 1 week after the application,conjunctival redness (score 2), chemosis (score 2) and whitish discharge (score 3), corneal opacity (score 2, area 4) were noted in the rabbit.At 2 weeks after the application,conjunctival redness (score 1), chemosis (score 1), discharge (score 2) and corneal opacity (score 3, area 4) were noted in the rabbit. The cornea was more bulging than the control eye. Importantly, the pupil did not give any response for light and it was impossible to fully examine the Iris at this time point. After discussion with the veterinarian, it was clear that the process of eye damage was irreversible. During the experiment, the control eye of the animal was symptom-free.

 

CONCLUSION

The test item, applied to rabbit eye mucosa, caused significant conjunctival and corneal irritant effects. Due to the local effects, the animal was euthanised for reasons of animal welfare and the study was terminated 2 weeks after the treatment. No further test was deemed necessary.

Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
mean score
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal 4397 (male)
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
mean score
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Remarks on result:
other: animal 4397 (male)
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Remarks:
mean score
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not fully reversible within: 2 weeks
Remarks on result:
other: animal 4397 (male)
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
mean score
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
not fully reversible within: 2 weeks
Remarks on result:
other: animal 4397 (male)
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion in vitro

The key study was performed using a reconstructed human epidermis model. EPISKIN (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 an analogue test item was evaluated according to the OECD No. 431 guideline.

 

Disks of EPISKIN (SM) (two units) were treated with 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 (NSC living) 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 to the analogue test item, the mean cell viability was 79.0% compared to the negative control (after adjustment for non-specific MTT reduction). 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.

Skin irritation in vitro

The key study was performed using a reconstructed human epidermis model. EPISKIN (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 irritation potential of an analogue test item was evaluated according to the OECD No. 439 guideline.

 

Disks of EPISKIN (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 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 (NSC living) from the test item. For each treated tissue, the viability was expressed as a % relative compared to the negative control. If the mean relative viability after 15 minutesexposure and 42 hours post incubation is ≤ 50 % of the negativecontrol, the test item is considered to be irritant to skin.

 

Following exposure to the analogue test item, the mean cell viability was 6.6 % compared to the negative control. This is below the threshold of 50%, therefore the test item was considered as being irritant to skin (removal of test item at 15 minutes was not fully achieved due to the physical characteristics of the test item). The experiment met the validity criteria, therefore the study was considered to be valid.

Skin irritation in vivo

A key acute skin irritation study involving an analogue test item was performed on New Zealand White rabbits in accordance with OECD Guidelines for Testing of Chemicals 404 (28th July 2015), Commission Regulation (EC) No 440/2008, B.4 (L 142, 30 May 2008) and OPPTS 870.2500 (EPA 712-C-98-196) August 1998.

 

A volume of 0.5 mL of the test item was applied to the skin of the experimental animals. The test items were applied as a single dose. Sterile gauze pads were placed on the skin of rabbits. These gauze pads were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The trunk was wrapped in clear plastic with medical tubing used to hold the patch in place. The untreated skin of each animal served as control. After the application, the remaining test item was removed by gently washing with Unguentum hydrophilicum nonionicum Ph. Hg. (washbasin ointment) and body temperature water. The treated skin surfaces were examined at 3 minutes and/or 1, 24, 48, 72 hours, 1 and 2 weeks after the patch removal. Additional general examinations were performed daily.

 

There was no mortality during the observation period and no test item-related clinical signs or effects on body weight. For animal 1, at 48, 72 hours, 1 and 2 weeks after patch removal, very slight erythema (score 1) was observed. For animal 2, at 24, 48, 72 hours, 1 and 2 weeks after patch removal, very slight erythema (score 1) was observed. At the 1-week observation, dry and cracked skin was also noted. For animal 3, at 24, 48, 72 hours, 1 and 2 weeks after patch removal, very slight erythema (score 1) was observed. At the 1-week observation, dry and cracked skin was also noted. The observed erythema did not reverse in any of the three animals during the 14-day observation period. Based on the scheme devised by Draize (1959), the analogue test item was considered to be a mild irritant.

Eye damage/irritation in vitro

A key study was performed on an analogue test item using isolated chicken eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 (26 July 2013).

 

After the zero reference measurements, the eye was held in horizontal position and approximately 30 μL of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 μL Benzalkonium chloride 5% solution. The negative control eye was treated with 30 μL of physiological saline (9% (w/v) NaCl solution). Examinations in Experiment I were three test item treated eyes, three positive control treated eyes and one negative control treated eye. In Experiment II, three test item treated eyes with more excessive rinsing, three positive control treated eyes, one negative control treated eye and one negative control treated eye with more excessive rinsing were examined. The observation time was also extended in Experiment II.

 

Results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

 

Test item was stuck on all cornea surfaces (confluent layer) during the full post treatment period and could not be rinsed off (all three cornea surfaces were not cleared at 240 minutes after the post-treatment rinse) and, as a result, the corneal opacity, the corneal swelling and the fluorescein retention could not be properly evaluated.

 

Due to the equivocal results, an additional experiment was performed for clarification as agreed by the sponsor.The experimental conditions were the same as in the first test, except for more excessive rinsing with physiological saline (31.7 °C) and an extension of the observation time. Moreover an additional control (standard controls and one negative control with similar excessive rinsing) demonstrated that the additional washing had no adverse effect.

 

Experiment I: Severe corneal swelling was observed during the four-hour observation period on test item treated eyes. Cornea opacity change could not be evaluated. Moderate fluorescein retention change (severity 2) was noted on three eyes. Test item was stuck on all cornea surfaces (confluent layer) after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

 

Experiment II: Moderate corneal swelling was observed during the five-hour observation period on test item treated eyes. Moderate cornea opacity change (severity 1 or 2) was observed on three eyes. Severe fluorescein retention change (severity 2 or 3) was noted on three eyes. These scores situate the test item as below, but close to, the cut-off for a severe irritant. Relatively large areas of the test item could be rinsed off, but small areas of the test item had material still stuck on all cornea surfaces even with after the post- excessive rinsing. All three cornea surfaces were not cleared fully at 300 minutes after the post-treatment rinse. The areas of test item stuck to the cornea could mask corneal effects underneath the particles and could also potentially result in mechanical damage in vivo.

 

The analogue test item was not classified as a severe irritant and not classified as non-irritant using the in vitro isolated chicken eye test method. It was therefore concluded that an in vivo study was required.

Eye damage/irritation in vivo

An acute eye irritation study involving an analogue test item was performed in the New Zealand White rabbit. The irritation effects of the test item were evaluated according to the Draize method (OECD No 405, 2012). One animal was used and the rabbit was treated with analgesic and anaesthetic as per the regulatory guideline.

 

The test item was placed into the conjunctival sac of the left eye of the animal. A single amount of 0.1 mL of the test item was administered as a single dose and, as a result of ocular effects observed, the treated eye was rinsed with physiological saline at one hour and 24 hours after the application of test item. The untreated right eye served as control. The eyes were examined at 1, 24, 48, 72 hours, 1 and 2 weeks after application of the test item.

 

The general state and behaviour of the animal were normal throughout the study period. The body weights of the rabbit were considered to be within the normal range of variability. No Initial Pain Reaction (IPR) was observed in the animal that was exposed to the test item. At subsequent time points no pain reaction (PR) was observed in this animal. At one hour after the application, conjunctival redness (score 2), chemosis (score 2), conjunctival discharge (score 3) and corneal opacity (score 2, area 4) were noted in the rabbit. The test item remained in the eye sac.At 24 hours after the application, conjunctival redness (score 3), chemosis (score 3), yellowish-white discharge (score 3), corneal opacity (score 2, area 4) and were noted in the rabbit.At 48 and 72 hours after the application, conjunctival redness (score 3), chemosis (score 3), yellowish-whitish and reddish discharge (score 3), corneal opacity (score 2, area 4) and were noted in the rabbit. At 1 week after the application, conjunctival redness (score 2), chemosis (score 2) and whitish discharge (score 3), corneal opacity (score 2, area 4) were noted in the rabbit. At 2 weeks after the application,conjunctival redness (score 1), chemosis (score 1), discharge (score 2) and corneal opacity (score 3, area 4) were noted in the rabbit. The cornea was more bulging than the control eye. Importantly, the pupil did not give any response for light and it was impossible to fully examine the Iris at this time point. After discussion with the veterinarian, it was clear that the process of eye damage was irreversible. During the experiment, the control eye of the animal was symptom-free.

 

The analogue test item, applied to rabbit eye mucosa, caused significant conjunctival and corneal irritant effects. Due to the local effects, the animal was euthanised for reasons of animal welfare and the study was terminated 2 weeks after the treatment. No further test was deemed necessary.

Justification for classification or non-classification

Skin corrosion: An analogue test item was considered to be non-corrosive to skin because cell viability was determined by OECD 431 (EPISKIN (SM) model) to be ≥ 35 % after 4 hours. Classification for skin corrosivity in accordance with Regulation (EC) No. 1272/2008 is therefore not required.

 

Skin irritation: An analogue test item was determined by OECD 439 (EPISKIN (SM) model) to be irritant to skin because percentage tissue viability after exposure and post-treatment incubation was found to be 50 %. These data were confirmed in vivo because, although mean scores from 24, 48 and 72-hour observations were zero for oedema and < 2.3 for erythema, non-reversibility was shown by erythema scores of 1 in all three animals after 7 and 14 days(OECD 404, EU Method B.4 and OPPTS 870.2500). Classification of the substance for skin irritation (category 2) is therefore applicable under the terms of Regulation (EC) No 1272/2008 and subsequent amendments.

Eye damage/irritation: In vitro test results did not permit a conclusive classification decision. However, when applied to the subsequently irrigated left eye of one rabbit, an analogue test item produced individual mean scores at 24/48/72 hours of 2 for corneal opacity, 0 for iritis, 3 for conjunctival redness and 4 for chemosis. The corneal opacity was also observed to worsen during a two-week observation period (final score 3), the ocular effects were declared irreversible after veterinary consultation, and the animal was sacrificed. Classification of the substance for serious eye damage (category 1) is therefore applicable under the terms of Regulation (EC) No 1272/2008 and subsequent amendments.