Registration Dossier

Administrative data

Description of key information

Skin corrosion in vitro: The test item was determined to be non-corrosive using the EPISKIN (SM) model (OECD 431).

 

Skin irritation in vivo: The test item was determined to be non-irritant (OECD 404, EU Method B.4 and OPPTS 870.2500).

 

Eye damage/irritation in vitro: The test item was determined to be non-irritant using the in vitro isolated chicken eye test method (OECD 438).

 

Eye damage/irritation in vivo: The test item was determined to be non-irritant (OECD 405, EU Method B.5 and OPPTS 870.2400).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 October 2016 to 14 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:
skin obtained from plastic surgery from multiple donors
Source strain:
other: adult
Vehicle:
physiological saline
Details on test system:
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.
- EPISKIN (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).

TEST SYSTEM
- Human skin: EPISKIN (SM) (Manufacturer: SkinEthic, France, Batch No.: 16-EKIN-041, Expiry Date: 17 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.
- For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.:16-EKIN-023, Expiry Date: 13 June 2016) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water
was discarded and the dead epidermis units were frozen on 10 June 2016 the frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Maintenance Medium). Further use of killed tissues was similar to living tissues.
- 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 069; Exp. Date: 19 October 2016); Flask of sterile “Assay Medium” (Batch No.: 16 ESSC 044; Exp. Date: 19 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.
- Furthermore, as the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used in. To avoid a possible double correction for colour interference, for non-specific colour in killed tissues, two additional disks were used.

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 10 mg 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, purple colour precipitate in the mixture was detected; therefore additional controls were used 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.
- As the test item was showed being an MTT-interacting substance and the test item had an intrinsic colour a third set of controls were necessary. The test item may bind to both living and killed tissues and therefore the NSMTT control may not only correct for potential direct MTT reduction by the test chemical, but also for colour interference arising from the binding of the test chemical to killed tissues. This could lead to a double correction for colour interference since the NSCliving control already corrects for colour interference arising from the binding of the test chemical to living tissues. To avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled) was needed to be performed.
- Therefore, in addition to the normal procedure, two additional test item-treated living tissues and two additional test item-treated killed tissues were used for the non-specific OD (optical density or absorbance) 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 (20 mg) was applied evenly to the epidermal surface of each of two test units.
- Physiological saline (100 μL) was then added to the test item to ensure good contact with the epidermis.
- 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 (23.2 to 25.5°C) covered with the plate lids.
- Note: The negative and positive controls were also part of a concurrent study (CiToxLAB study code: 16/230-039B) performed in the same experimental period using the same batch of chemicals and same batch of skin units.
- As the test item was showed being an MTT-interacting substance, in addition to the normal procedure, two test item treated killed epidermis and two negative control treated killed epidermis were used in the study (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues
was different than the batch of the living tissues. The same treatment steps were followed in case of the killed tissues as in case of the living tissues.

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.

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 as described above.

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 (PCraw) 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:
20 mg of test item
Duration of treatment / exposure:
4 hours
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
Two
Irritation / corrosion parameter:
% tissue viability
Value:
107.7
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 0.002.
- The Non Specific Colour % (NSC living %) was calculated as 0.2 % (see Table 1, attached). This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.
- As colour change (purple precipitate) was observed after three hours of incubation of the test item in MTT working solution, thus the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Results of the additional controls on killed epidermis are shown in Table 2 (attached). Based on these observed mean OD (0.008), the calculated NSMTT% is 1.1%.
- As the test item was showed being an MTT-interacting substance and the test item had an intrinsic colour, two additional test item-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.008, Non Specific Colour % (NSCkilled%) was calculated as 1.1% (see Table 3, attached). Because the NSCliving% was not used, therefore correction with NSCkilled% 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 4 (attached).
- The mean OD value for the test item treated skin samples showed 107.7 % 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 (0.780).
- The positive control treated tissues showed 0.8 % 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 6.0 %.
- The mean OD value of the blank samples (acidified isopropanol) was 0.047.
- 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:
Results from the in vitro EPISKIN (SM) indicate that the test item is 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. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. 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 107.7 % 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 irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 November 2016 to 24 November 2016
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 deviations with no impact on integrity or results of the study (see below)
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
various deviations with no impact on integrity or results of the study (see below)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Deviations:
yes
Remarks:
various deviations with no impact on integrity or results of the study (see below)
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
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.
- Number of animals: 3
- Sex: Male
- Age of animals at treatment: 16 weeks old
- Body weight range at the beginning of the in-life phase: 3608 – 4009 g
- Body weight range at the end of the in-life phase: 3820 – 4071 g
- Date of receipt: 28 September 2016
- Acclimatisation time: 43 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
- Animal health: Only animals in acceptable health condition were used for the test.
- Number of animal room: 032
- 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: 20.45 - 25.04 °C
- Relative humidity: 28.05 – 68.13%
- Ventilation: 15-20 air exchanges/hour
- Temperature and relative humidity values were measured continuously.

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: 0003491507, expiry date: 26 October 2016
(ii) Batch number: 0003556255, expiry date: 23 November 2016
(iii) Batch number: 0003659483, expiry date: 03 January 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.
Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
0.5 g
Duration of treatment / exposure:
4 hours
Observation period:
14 days
Number of animals:
Three
Details on study design:
MATERIALS USED FOR TREATMENT
Insuline syringe (1mL)
Batch number: 150502
Expiry date: 31 May 2020
Manufacturer: Kanghua Co. Ltd.

Pharmico steril mull-lap (sterile gauze pads)
Batch number: 1510675
Expiry date: 30 November 2020
Manufacturer: Dispomedicor Zrt.
GSM Betasilk (surgical silk tape)
Batch number: 11122334
Expiry date: 31 December 2016
Manufacturer: GS Medical Ltd.

Ramofix (flexible bandage)
Batch number: 2016 JAN 11
Expiry Date: 29 January 2021
Manufacturer: Ramofix Trade Kft.

MATERIALS USED FOR EUTHANASIA
Primazin (Xylazin 2%)
Batch No.: 1505130-03
Expiry date: 31 May 2017
Manufacturer: Alfasan International B.V.

Ketamidor (Ketamin 10%)
Batch number: 0914489 AG
Expiry date: 31 August 2017
Manufacturer: Richterpharma ag

Euthanimal 40% (pentobarbital sodium)
Batch number: 1409236-06
Expiry date: 30 September 2017
Manufacturer: AlfasanNederland B.V.

DOSAGE
- The test item was applied as a single dose of 0.5 g to the test area.
- Sufficient water was added to dampen the material to ensure good contact with the skin.
- 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.
- The test item was applied to an approximately 6 cm² area of intact skin as follows:
(i) A single layer of a fine medical gauze (open-weave with large holes) of approximately 5x5 cm was placed over the application area,
(ii) The appropriate amount of test item was carefully spread over the application area (the gauze helped maintain the test item in place),
(iii) Three more layers of gauze were placed over the test item,
(iv) These gauze patches were kept in contact with the skin by a patch of clear plastic with a surrounding adhesive hypoallergenic plaster to ensure continued good contact between the moistened test item and the shaved skin.
(v) The entire trunks of the animals were wrapped with plastic wrap for 4 hours.
(vi) Medical elastic tubing was placed over the plastic to keep it in place.
- An initial test was performed using one animal. One hour after application of the test item, the application site was examined. No severe irritation or corrosive effect was found in the initial test, therefore the bandage was replaced and the exposure continued for a further 3 hours (a total 4 hours exposure). Two additional animals were then included in the study.

DURATION OF EXPOSURE
- Duration of exposure: 4 hours.
- After the treatment period, the test item was removed with water at body temperature.

CLINICAL OBSERVATIONS
- Animals were examined for signs of erythema and oedema, and the responses scored at 60 minutes and then at 24, 48, 72 hours, 7 days and 14 days 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).
- The animals were observed for 72 hours (2 animals) or for 2 weeks (1 animal) and the duration of the study was sufficient to evaluate fully the reversibility or irreversibility of the effects observed.

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 animals 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:
male
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Remarks on result:
other: animal 2739
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: animal 2732
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
male
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 14 d
Remarks on result:
other: animal 2728
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: animal 2739
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: animal 2732
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: animal 2728
Irritant / corrosive response data:
- Erythema scores for each animal are given in Table 1 (attached).
- Oedema scores for each animal are shown in Table 2 (attached).
- There was no mortality observed during the study.
- There was no test item related effect on body weight.
- No test item related clinical signs were noted.
- At 1 hour after patch removal, no clinical signs were observed on the skin of the treated animals.
- At 24 hours after patch removal, very slight erythema was observed in two animals.
- At 48 and 72 hours after patch removal, only one animal had very slight erythema, therefore the observation of the other two animals were terminated after 72-hour observation.
- At 7 days after patch removal, the animal still had very slight erythema on the skin.
- At 14 days after patch removal, the remaining animal became also symptom free and thus the study was terminated.
- The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema were 0.33, 0.00 and 1.00.
- The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for oedema were 0.00, 0.00 and 0.00.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item caused very slight irritation on the skin of two animals out of the three, which was reversible within 2 weeks.
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

The test item had previously been shown to be non-corrosive in vitro and irritancy of the test item was evaluated according to the Draize A volume of 0.5 g of test item was applied to the skin of the experimental animals. The test item was applied as a single dose. Sufficient water was added to dampen the material to ensure good contact with the 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 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. Parameters monitored during this study included mortality, body weight measurements and clinical observations. method. After 4 hours, the remaining test item was removed with water at body temperature. To assess skin irritation, the three animals were examined at 1, 24, 48, 72 hours,

then one of the rabbits at 7 and 14 days after the patch removal. Additional general examinations were performed daily.

 

RESULTS

There was no mortality during the observation period and no test item related effect on body weight. One hour after patch removal, no clinical signs were observed on the skin of

the treated animals. At 24 hours after patch removal, very slight erythema was observed in two animals. At 48 and 72 hours after patch removal, only one animal had very slight erythema, therefore the observation of the other two animals were terminated after 72-hour observation. At 7 days after patch removal, the animal still had very slight erythema on the skin. At 14 days after patch removal, the remaining animal became also symptom free and thus the study was terminated. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema were 0.33, 0.00 and 1.00. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for oedema were 0.00, 0.00 and 0.00.

 

CONCLUSION

The test item caused very slight irritation on the skin of two animals out of the three, which was reversible within 2 weeks.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
03 October 2016 to 11 November 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
GLP compliance:
yes (incl. certificate)
Species:
chicken
Strain:
other: COBB 500 in Experiment I and ROSS 308 in Experiment II
Details on test animals or tissues and environmental conditions:
- Source: TARAVIS KFT, 9600 Sárvár, Rábasömjéni út.129., Hungary
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 mg
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 has been recognised as a valuable alternative to the Draize eye irritation test regarding ocular corrosivity or severe eye irritancy testing, because it represents a test system nearest to the in vivo test, without the need to use live animals. In the Isolated Chicken Eye Test (ICET) the
test compound is applied in a single dose onto the cornea of isolated eyes, which are obtained from slaughter animals.
- 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).

TEST ITEM SOLUBILITY AND FORMULATION
- The solubility of the test item in physiological saline was tested prior to the experiment (30 mg test material in 1 mL physiological saline). The test item did not dissolve in physiological saline.
- The test item was applied in its original form (although the test item was pulverized into smaller pieces).

SUBSIDIARY MATERIAL
- Fluorescein 10 % (w/v) (Alcon; Lot 250817F; Expiry date 31 May 2017; Room temperature storage conditions).
- This material was mixed with physiological saline (Manufacturer: B. Braun Pharmaceuticals SA, Lot number: 61461Y05-1, Expiry date: March 2019) to achieve the final concentration of 2% (w/v).
- The resulting solutions were stored at room temperature (Dispensary code: S43108, Expiry date: 12 October
2016 in Experiment I; Dispensary code: S43111, Expiry date: 24 November 2016 in Experiment II).

CHICKEN HEADS COLLECTION AND TRANSPORT
- 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 were 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.
- Test item (30 mg) was applied was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
- In each experiment, the negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered imidazole.
- One eye was treated with physiological saline, three eyes with the test item and another three eyes with powdered imidazole in each experiment.

TEST ITEM REMOVAL
- The time of application was noted, 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 solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.
- Additional gentle rinsing with 20 mL saline was performed after treatment and at each time point when the test item or positive control material remaining on the cornea was observed.
- The test item treated eyes (where the test item was stuck on the cornea surfaces) were rinsed additional gentle rinsing at least 2x20 mL saline after treatment in each experiment.

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: KTM15711, Expiry date: April 2018) 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: 3 x I
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: 3 x I
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.
- The test item showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD no. 438 guideline. - The second experiment confirmed the negative results. However, test item stuck on two cornea surfaces at 240 minutes after the post-treatment rinse in the first experiment. Based on our experience for some cases where test item adheres to the cornea in vitro, this can result in severe irritation in vivo (probably due to mechanical effects of abrasive particles stuck to the cornea). However, in this case only a minimal amount of test item was observed by the end of the observation period, thus this fact was considered not to adversely affect the results and study interpretation. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes, the test item was
non-irritant, UN GHS Classification: No Category.
- 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 control (Imidazole) was 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 and 1.6 (see Appendix 1, attached).
- The negative control Physiological saline was classified as non-irritating, UN GHS Classification: Non-classified. (see results for Experiments 1 and II, attached).

VALIDITY OF THE TEST
- Historical control data are attached.
- The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the in vitro eye irritation assays in isolated chicken eyes, the test item was determined to be non-irritant, UN GHS Classification: No Category.
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 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

 

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.

 

As the test item was solid, the observed negative result of the first experiment was confirmed by a second experiment.

 

Experiment I: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. Cornea opacity change (severity 0.5) was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on one eye. Minimal amount of test item was stuck on two cornea surfaces at 240 minutes after the post-treatment rinse.

 

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. Cornea opacity change (severity 0.5) was observed on one eye. Fluorescein retention change (severity 0.5) was noted on three eyes.

 

CONCLUSION

Based on the in vitro eye irritation assays in isolated chicken eyes, the test item was determined to be non-irritant, UN GHS Classification: No Category.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 December 2016 to 18 December 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:
humidity outside expected range with no impact on integrity or results of the study (see below)
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
humidity outside expected range with no impact on integrity or results of the study (see below)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
yes
Remarks:
humidity outside expected range with no impact on integrity or results of the study (see below)
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
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 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. Both eyes of each 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: 3 animals
- Age of animals at treatment: 10-11 weeks old
- Sex: Male
- Body weight range on the day of treatment: 2697 g – 2865 g
- Body weight range before euthanasia: 2739 g – 2911 g
- Date of receipt: 07 December 2016
- Acclimatisation time: 6/8 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
- Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
- 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.
- Number of animal room: 032
- Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- Temperature: 19.54 – 21.69 °C
- Relative humidity: 29.56 – 55.82 %
- 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. Animals were provided with the following batches:
(i) Batch number: 0003659483, expiry date: 03 January 2017
(ii) Batch number: 0003801145, expiry date: 01 March 2017
- 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 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.
Amount / concentration applied:
0.1 g
Duration of treatment / exposure:
72 hours (the treated eye was rinsed with physiological saline at the one hour observation but test item remained in the eye sac)
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
Three
Details on study design:
MATERIALS USED FOR WASHING
Disposable Syringe Luer Solo, 20 mL
Batch number: 3M09048
Expiry date: 31 December 2018
Manufacturer: B. Braun Melsungen AG

Saline (0.9% NaCl)
Batch number: 61461Y05-1
Expiry date: 31 March 2019
Manufacturer: B. Braun Melsungen AG

SYSTEMIC OPIATE ANAESTHETIC
Bupaq (0.3 mg/mL buprenorphine)
Batch number: 0115034AD
Expiry date: 31 December 2017
Manufacturer: Richterpharma AG

TOPICAL OCULAR ANAESTHETIC
Humacain (4 mg/mL oxybuprocaine)
Batch number: 0470515
Expiry date: 31 May 2018
Manufacturer: Teva Pharmaceuticals Ltd.

NON-STEROIDAL ANTI-INFLAMMATORY DRUG
Meloxidyl (5 mg/mL meloxicam)
Batch number: T01084
Expiry date: 30 November 2018
Manufacturer: Ceva Santé Animale

MATERIALS USED FOR EUTHANASIA
Ketamidor (100 mg/mL ketamine)
Batch number: 0914489AG
Expiry date: 31 August 2017
Manufacturer: Richterpharma AG

Primazin (2% xylazin)
Batch number: 1505130-03
Expiry date: 31 May 2017
Manufacturer: Alfasan International B.V.

Euthanimal (40% pentobarbital sodium)
Batch number: 1409236-06
Expiry date: 30 September 2017
Manufacturer: Alfasan International B.V.

IDENTIFICATION OF pH
- The pH of the test item was measured as 5.0, permitting its use in the animal study.

IN VITRO STUDY RESULTS
- An in vitro eye irritation assay in isolated chicken eyes with the test item was performed by CiToxLAB Hungary Ltd. Based on the results of this test, the substance is non-irritant and it was concluded that an in vivo study could be performed for classification purposes.

PRE-STUDY EXAMINATION
- Three male animals in acceptable health condition were 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
- Initially only one rabbit was treated with test item.
- The local effects were non-irritant (all scores were zero) at 24 hours, then two further rabbits were treated with the test item.

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 subcutaneous injection (SC) under direct Veterinary supervision. The systemic opiate analgesic was again injected approximately 12 hours after the post-treatment analgesic.
- Systemic opiate analgesic: Buprenorphine 0.01 mg/kg.
- Topical ocular anaesthetic: Humacain (oxybuprocaine) one-two drops per eye.
- Nonsteroidal anti-inflammatory drug: Meloxicam 0.5 mg/kg.

ADMINISTRATION OF TEST ITEM
- Test item (0.1 mL) was administered to the animals.
- The test substance was placed in the conjunctival sac of the left eye of each 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 test item was supplied as a solid, however due to the nature of the test item it was not possible to grind it. The fine dust was prepared for treatment with scraping of the surface of the supplied material.
- The untreated contralateral eye served as the control.
- As the solid test item remained in the eye sac in all animals at the one hour observation time point, the treated eye of test animals was rinsed with physiological saline solution.

OBSERVATIONS AND SCORING
- The eyes were examined at 1, 24, 48, 72 hours after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes.
- Any clinical signs of toxicity or signs of ill-health during the study were recorded.
- All rabbits were examined for distress at least twice daily, with observations at least 6 hours apart. Clinical observations or signs of ill-health 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 (Euthanimal 40 %). 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 3, attached).
Irritation parameter:
cornea opacity score
Basis:
other: all animals
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: animals 6192, 6201 & 6203
Irritation parameter:
iris score
Basis:
other: all animals
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Remarks on result:
other: animals 6192, 6201 & 6203
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
other: all animals
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
other: animals 6192, 6201 & 6203
Irritation parameter:
chemosis score
Basis:
other: all animals
Remarks:
male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
other: animals 6192, 6201 & 6203
Irritant / corrosive response data:
EXAMINATION OF EYE IRRITANCY
- No Initial Pain Reaction (IPR) or any Pain Reaction (PR) was observed during the experimental period.
- Animal 1 (6192) clinical observation: At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 2) were noted in the rabbit. Test item remained in the eye sac. At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.
- Animal 2 (6201): At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 2) were noted in the rabbit. Test item remained in the eye sac. At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.
- Animal 3 (6203): At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 2) were noted in the rabbit. Test item remained in the eye sac. At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.
- As no clinical signs were observed, the study was terminated after 72 hours observation.
- During the study, the control eye of each animal was symptom-free.
- The general state and behaviour of animals were normal throughout the study period.
- Individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) for chemosis, discharge, redness, cornea and iris were were 0.00 in all three animals.
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 3, attached).

GENERAL DAILY EXAMINATION
- There were no clinical signs observed that could be related to treatment.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 24 hours.
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 in accordance with OECD Guidelines for Testing of Chemicals 405 (2nd October 2012), Commission Regulation (EC) No 440/2008, B.5 (L 142, 30 May 2008) and OPPTS 870.2400 (EPA 712-C-98-195) August 1998.

 

METHODS

Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Three animals were used to make the classification. The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A single amount of 0.1 g of the test item was administered as a single dose. The eyes were examined at 1, 24, 48 and 72 hours after application.

 

RESULTS

No Initial Pain Reaction (IPR) or any Pain Reaction (PR) was observed during the experimental period.

 

At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 2) were noted in Animal 1 (No: 6192). Test item remained in the eye sac.

 

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed. At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 2) were noted in Animal 2 (No: 6201). Test item remained in the eye sac.

 

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed. At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 2) were noted in Animal 3 (No: 6203). Test item remained in the eye sac.

 

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed. As no clinical signs were observed, the experiment was terminated after 72 hours observation. During the experiment, the control eye of each animal was symptom-free.

 

The general state and behaviour of animals were normal throughout the study period. No mortality occurred during the study. The bodyweights of all rabbits were considered to be within the normal range of variability. Individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) for chemosis, discharge, redness, cornea and iris were were 0.00 in all three animals.

 

CONCLUSION

The test item, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 24 hours.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion in vitro

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.

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. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSC killed), two additional disks were used. 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 test item, the mean cell viability was 107.7 % 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. Results from the in vitro EPISKIN (SM) indicate that the test item is non-corrosive to skin.

Skin irritation in vitro

An in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available.

Skin irritation in vivo

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.

The test item had previously been shown to be non-corrosive in vitro and irritancy of the test item was evaluated according to the Draize. Test item (0.5 g) was applied to the skin of the experimental animals. The test item was applied as a single dose. Sufficient water was added to dampen the material to ensure good contact with the 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 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. Parameters monitored during this study included mortality, body weight measurements and clinical observations. method. After 4 hours, the remaining test item was removed with water at body temperature. To assess skin irritation, the three animals were examined at 1, 24, 48, 72 hours, then one of the rabbits at 7 and 14 days after the patch removal. Additional general examinations were performed daily.

There was no mortality during the observation period and no test item related effect on body weight. One hour after patch removal, no clinical signs were observed on the skin of the treated animals. At 24 hours after patch removal, very slight erythema was observed in two animals. At 48 and 72 hours after patch removal, only one animal had very slight erythema, therefore the observation of the other two animals were terminated after 72-hour observation. At 7 days after patch removal, the animal still had very slight erythema on the skin. At 14 days after patch removal, the remaining animal became also symptom free and thus the study was terminated. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema were 0.33, 0.00 and 1.00. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for oedema were 0.00, 0.00 and 0.00. The test item caused very slight irritation on the skin of two animals out of the three, which was reversible within 2 weeks.

Eye irritation in vitro

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).

 

After the zero reference measurements, the eye was held in horizontal position and 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

 

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.

 

As the test item was solid, the observed negative result of the first experiment was confirmed by a second experiment.

Experiment I: No significant corneal swelling (mean 5%) was observed during the four-hour observation period on test item treated eyes. Cornea opacity change (severity 0.5) was observed on three eyes. Fluorescein retention change (severity 0.5) was noted on one eye. Minimal amount of test item was stuck on two cornea surfaces at 240 minutes after the post-treatment rinse.

 

Experiment II: No significant corneal swelling (mean 5%) was observed during the four-hour observation period on test item treated eyes. Cornea opacity change (severity 0.5) was observed on one eye. Fluorescein retention change (severity 0.5) was noted on three eyes.

Based on the in vitro eye irritation assays in isolated chicken eyes, the test item was determined to be non-irritant, UN GHS Classification: No Category.

Eye irritation in vivo

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 in accordance with OECD Guidelines for Testing of Chemicals 405 (2nd October 2012), Commission Regulation (EC) No 440/2008, B.5 (L 142, 30 May 2008) and OPPTS 870.2400 (EPA 712-C-98-195) August 1998.

 

Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Three animals were used to make the classification. The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A single amount of 0.1 g of the test item was administered as a single dose. The eyes were examined at 1, 24, 48 and 72 hours after application.

 

No Initial Pain Reaction (IPR) or any Pain Reaction (PR) was observed during the experimental period.

At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 2) were noted in Animal 1 (No: 6192). Test item remained in the eye sac.

 

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed. At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 2) were noted in Animal 2 (No: 6201). Test item remained in the eye sac.

 

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed. At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 2) were noted in Animal 3 (No: 6203). Test item remained in the eye sac.

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed. As no clinical signs were observed, the experiment was terminated after 72 hours observation.

 

During the experiment, the control eye of each animal was symptom-free. The general state and behaviour of animals were normal throughout the study period. No mortality occurred during the study. The bodyweights of all rabbits were considered to be within the normal range of variability. Individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) for chemosis, discharge, redness, cornea and iris were 0.00 in all three animals.

 

The test item, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 24 hours.

Justification for classification or non-classification

Skin corrosion/irritation: The 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 corrosion is therefore unnecessary under the terms of Regulation 1272/2008 and subsequent amendments. When investigated in vivo, mean scores (24/48/72 hours) in the rabbit were < 2.3 for erythema and zero for oedema and the effects were fully reversible. Classification for skin irritation is therefore unnecessary under the terms of Regulation 1272/2008 and subsequent amendments.

 

Eye damage/irritation: The test item was classified as non-irritant using the in vitro isolated chicken eye test method because ICE classes for corneal swelling, opacity and flurorescein retention were all determined to be I (OECD 438). When investigated in vivo, mean scores (24/48/72 hours) in the rabbit were zero for corneal opacity, iritis, conjunctival redness and chemosis. Classification for eye irritation is therefore unnecessary under the terms of Regulation 1272/2008 and subsequent amendments.