2,2,2,4,4,4,4-heptakis[(2-ethylhexanoyl)oxy]cyclodimolybdoxan-2-yl 2-ethylhexanoate

Endpoint summary

Description of key information

HCAT was demonstrated to be non-irritant to the skin (no category) when tested in the EpiSkin® in vitro irritation assay OECD Guideline 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.  HCAT was demonstrated to be an eye irritant when tested in vitro using the SkinEthic® HCE reconstructed human corneal model.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 2011 to 08 September 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted according to International Guidelines and in accordance with the OECD Principles of Good Laboratory Practice.

Qualifier:

according to guideline

Guideline:

other: OECD (2010), In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, OECD Guidelines for the Testing of Chemicals No. 439, OECD, Paris.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on study design:

MTT Direct Reduction Test (Preliminary Assay)
Prior to conducting the irritation assay, it was first necessary to determine if HCAT was capable of reducing MTT to its formazan metabolite. Since the assay depends on the ability of viable skin cells to reduce MTT to formazan, any MTT reduction caused by the test item would interfere with the integrity of the results. Three replicate samples (10 μL) of HCAT were added to a solution of MTT in PBS (2 mL, 0.3 mg/mL). Samples were then incubated for 3 h in a humidified incubator set to maintain temperature and CO2 levels of 37°C and 5%, respectively. Production of purple-coloured formazan was assessed by visual inspection. Three replicates of the positive control (eugenol, 10 μL) and the negative control (water, 10 μL) were tested in parallel to demonstrate the efficacy of the MTT solution.

Application of Test Items and Controls
Aliquots (10 μL) of HCAT, the positive control (5% SDS) and the negative control (Dulbecco’s PBS) were applied to three replicate EpiSkin® tissues using a positive displacement pipette. The pipette tip was used to gently spread the test item and controls over the entire exposed area of the EpiSkin® tissues. The surface area of the EpiSkin® was 0.38 cm2, therefore the application rate was 26.3 μL/cm2.

Non-Viable Skin Controls
Due to the inconclusive result obtained in the MTT direct reduction test (and the physical nature of the test item), non-viable controls were tested in parallel with the irritation assay. Since any test item that was not removed by the washing procedure could affect the absorbance values of the formazan extractions, the controls were employed to quantify this effect. Aliquots (10 μL) of HCAT were applied to three replicate non-viable EpiSkin® tissues as described in Section 6.7. Since non-viable tissues can still retain some ability to reduce MTT, three undosed non-viable tissues were also incubated with the assay.

Terminal Exposure Procedures (15 min Post Dose)
After exposure to the tissues for 15 min, the test item was removed from the surface of the skin using a combination of Dulbecco’s PBS and tissue paper. Complete removal of the test item was not achieved due to its inherent stickiness and hydrophobicity (the PBS wash had little effect). The positive and negative controls were removed from the EpiSkin®surface by rinsing with Dulbecco’s PBS (25 mL). The EpiSkin® units were then blotted dry with tissue paper, transferred to fresh maintenance medium, and incubated for 42 h in a humidified incubator set to maintain temperature and CO2 levels of 37°C and 5%, respectively.

MTT Test
Set up
After the recovery period, all EpiSkin® units were transferred to a 12 well microtiter plate containing MTT solution in assay medium (0.3 mg/mL, 2 mL per well). Each unit was tapped gently on tissue paper to remove residual moisture before transferring to the MTT solution. The assay was then incubated for 3 h in a humidified incubator set to maintain temperature and CO2 levels of 37°C and 5%, respectively.

Formazan Extraction
After incubation, each skin unit was removed from the MTT solution and gently tapped on tissue paper to remove any residual moisture. The exposed skin was then completely removed from the RhE unit using a biopsy punch. The epidermis was separated from the collagen matrix and both layers added to a microcentrifuge tube containing acidified isopropanol (500 μL). Samples were then stored for 46.5 h, in a refrigerator set to maintain a temperature of 4°C.

Absorbance Measurement
The formazan extractions were removed from the refrigerator and duplicate aliquots of each sample (200 μL) added to a 96 well plate. Plates were analysed using an MRX plate reader using wavelength measurement at 550 nm. Absorbance values were calculated against the background acidified isopropanol sample reading.

CALCULATIONS
Optical Density (OD) OD Treated Tissue (TT) = OD TT raw – OD blank mean
OD Negative Control (NC) = OD NC raw – OD blank mean
The corrected mean OD Negative Control corresponds to 100% viability
OD Positive Control (PC) = OD PC raw – OD blank mean

Individual Viabilities (%)
% Positive Control 1 = [OD PC1 / Mean OD NC] x 100
% Positive Control 2 = [OD PC2 / Mean OD NC] x 100
% Positive Control 3 = [OD PC3 / Mean OD NC] x 100
% Treated Tissue 1 = [OD TT1 / Mean OD NC] x 100
% Treated Tissue 2 = [OD TT2 / Mean OD NC] x 100
% Treated Tissue 3 = [OD TT3 / Mean OD NC] x 100

Mean Viabilities (%)
Mean Positive Control % = (%PC1 + %PC2+ %PC3) / 3
Mean Treated Tissue % = (%TT1 + %TT2 + %TT3) / 3

Relative Viability (%)
% Viability = [ODTT / ODNC] x 100

Non-Specific MTT Reduction (NSMTT)
ODku: untreated killed tissues
ODkt: chemical treated killed tissues
NSMTT= [(ODkt - ODku) / ODNC] x 100

True MTT Metabolic Conversion (TODTT)
ODTV: chemical treated viable tissues
ODTT: true MTT metabolic conversion for treated tissue.
TODTT = [ODTV – (ODkt - ODku)]

True Relative Viability (%)
True % Viability = [TODTT / ODNC] x 100

Acceptance Criteria
The assay was deemed acceptable if the following criteria were met: The non-specific MTT reduction was ≤30% relative to the negative control optical density.
The mean OD value of the 3 negative control tissues was >0.6 and <1.5 and the Standard Deviation value (SD) of the % viability was <18%.
The mean % viability of the 3 positive control tissues was <30% and the SD was <18%.
The mean % viability SD of the 3 treated tissues was <18%.

Interpretation of Skin Irritancy
The test item was considered to be irritant to skin in accordance with GHS category 2 if the tissue viability after exposure and post-treatment incubation was less than or equal (≤) to 50%.
Depending on country/regional regulatory requirements, the test item may be considered to be “no category” if the tissue viability after exposure and post-treatment incubation was more than (>) 50%.

Data Presentation
Data presented in results, tables, figures and appendices are computer generated and rounded appropriately for inclusion in the report. As a consequence, calculation of values from data presented will, in some instances, yield minor variations.

PROTOCOL COMPLIANCE
Section 9.2.5 of the protocol states that the formazan extractions will be stored in a refrigerator set to maintain a temperature of 4°C for 72 ± 12 h. The actual storage time was 46.5 h. The storage time specified in the protocol was taken from the ECVAM SOP and does not represent an optimal extraction period but a practical one (i.e. weekend incubation). The 46.5 h extraction period is considered to be sufficient to ensure complete extraction of the formazan. Therefore, this deviation does not affect the integrity of the study.

Irritation / corrosion parameter:

other: other: % Viability of EpiSkin Tissues

Value:

60.24

Remarks on result:

other:

Remarks:

Basis: other: HCAT (Corrected). Time point: 42h recovery time. Max. score: 60.24. Reversibility: no data. Remarks: SD: 6.55 %; CV: 10.88 %. (migrated information)

Irritation / corrosion parameter:

other: other: % Viability of EpiSkin Tissues

Value:

14.49

Remarks on result:

other:

Remarks:

Basis: other: 5% SDS Solution (Positive Control). Time point: 42h recovery time. Max. score: 14.49. Reversibility: no data. Remarks: SD: 3.63 (%); CV: 25.05 %. (migrated information)

Irritation / corrosion parameter:

other: other: % Viability of EpiSkin Tissues

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: PBS Solution (Negative Control). Time point: 42h recovery time. Max. score: 100.0. Reversibility: no data. Remarks: SD: 7.20 %; CV: 7.2 %. (migrated information)

MTT Direct Reduction Test (Preliminary Assay)

The test was scored by visual assessment of the formation of purple-coloured formazan. The positive control (eugenol) reduced the MTT solution to purple-coloured formazan. The negative control (PBS) did not reduce MTT to formazan after 3 h incubation. Due to the deep red colour of HCAT, it was difficult to distinguish if it reduced the MTT. Therefore, non-viable skin controls were employed in the irritation assay to determine the effect of any unremoved test item on the viability results.

Negative Controls

The negative control results were similar for the three viable EpiSkin® units dosed with Dulbecco’s PBS. Exposure to Dulbecco’s PBS resulted in a mean EpiSkin® viability of 100.00% ± 7.20%. The results were within the acceptance criteria defined in the ECVAM validation SOP.

Positive Controls The positive control results were similar for the three viable EpiSkin® units dosed with SDS (5%, w/v). Exposure to SDS (5%, w/v) resulted in a mean EpiSkin® viability of 14.49% ± 3.63%. These results were within the acceptance criteria defined in the ECVAM validation SOP.

HCAT

Non-Viable Controls The mean absorbance value of the three replicate non-viable tissues dosed with HCAT was 0.309 ± 0.209. The mean absorbance value of the three replicate undosed non-viable tissues was 0.105 ± 0.027. Therefore, the absorbance value for the effect of the unremoved test item was 0.204. This value was subtracted from the absorbance value of the viable tissues dosed with HCAT.

Viable Tissues

The results were similar for the three viable EpiSkin® units dosed with HCAT. Exposure to HCAT resulted in a mean EpiSkin® viability (corrected for effect of unremoved test item) of 60.24% ±6.55 % of the negative control value.

Discussion

Due to the physical nature of HCAT, it was not possible to completely remove the test item by washing. Tissue swabs were employed to facilitate the wash procedure but it was apparent that some test item remained on the EpiSkin® surface. The effect of this unremoved test item on the absorbance values was calculated using non-viable controls and was subtracted from the absorbance results for the viable tissues dosed with HCAT. Despite the failure to completely remove the test item prior to the recovery period, the results of the assay are considered to be valid since the non-viable controls had visibly more HCAT remaining on the EpiSkin® surface after washing than the living cells. Therefore, the absorbance value for the effect of the test item on the viability results is considered to be worst case.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

HCAT was demonstrated to be non-irritant (no category) when tested in the EpiSkin® in vitro irritation assay.

Executive summary:

HCAT was tested in the SkinEthic EpiSkin® in vitro irritation assay. The endpoint of the assay was the estimation of cell viability by measuring the reduction of methylthiazoldiphenyl-tetrazolium bromide (MTT) to its formazan metabolite by mitochondrial reductase. Irritant materials were identified by their ability to reduce cell viability below a threshold of 50% of the negative control value. Prior to the conduct of the irritation assay, a preliminary test was conducted to assess the intrinsic ability of HCAT to reduce MTT to formazan. Due to the deep red colour of the test item, it was difficult to interpret the results of the test. Therefore, non-viable tissue controls were dosed in parallel with the irritation assay to quantify the effect of any unremoved test item on the absorbance results. The dermal irritation potential was assessed by applying HCAT (10 μL) to the exposed surface of three viable EpiSkin® reconstructed human epidermis (RhE) units for 15 min. The surface area of the EpiSkin® was 0.38 cm2, therefore the application rate was 26.3 μL/cm2. After the 15 min exposure period, the test item was washed from the surface of the EpiSkin® using Dulbecco’s phosphate-buffered saline (PBS) (25 mL) and tissue swabs. The EpiSkin® was then incubated for a recovery period of 42 h in a humidified incubator set to maintain temperature and CO2 levels of 37°C and 5%, respectively. The EpiSkin® units were then transferred to assay medium containing MTT (0.3 mg/mL) and returned to the incubator for 3 h. After incubation, biopsies of the EpiSkin® membranes were removed, added to acidified isopropanol and refrigerated for 46.5 h (protected from light) in order to extract the formazan. The formazan production (cell viability) was assessed by measuring the optical density of the extracts at a wavelength of 550 nm. Three replicates of the positive control, sodium dodecyl sulphate (5% SDS solution) (10 μL), and the negative control, PBS (10 μL) were tested in parallel to demonstrate the efficacy of the assay. The viability of each individual EpiSkin®tissue was calculated as a percentage of the mean negative control viability (defined as 100%). The HCAT absorbance results were corrected to account for the effect of any test item not removed by the wash procedure. Exposure to HCAT resulted in a mean EpiSkin® viability of 60.24% ±6.55 % of the negative control value. Exposure to the positive control, SDS, resulted in a mean EpiSkin® viability of 14.49% ± 3.63% of the negative control value. In conclusion, HCAT was demonstrated to be non-irritant (no category) when tested in the EpiSkin® in vitro irritation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 February 2011 to 13 July 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was conducted in accordance with ECVAM pre-validated alternative testing methodologies and in accordance with the OECD Principles of Good Laboratory Practice as incorporated into the United Kingdom (Statutory Instrument for GLP). While this study does not provide quantitative results to grade the level of irritation/corrosion that would be provided by certainin-vivo studies, it allows the application of a conservative human health classification for irritation/corrosion.

Principles of method if other than guideline:

The SkinEthic® HCE model assesses the corrosion/irritation potential of a test item by examining the cytotoxic effects of the test item after a defined exposure period and recovery time.
The endpoint of the HCE assay is the estimation of cell viability by assaying the reduction of MTT by mitochondrial enzymes. Corrosive/irritant materials are identified by their ability to reduce cell viability below a threshold of 50% of the negative control value.
The SkinEthic reconstructed Human Corneal Epithelium model has undergone ECVAM prevalidation testing and has been shown to have a high correlation with existing in vivo and in vitro data for known eye irritants and non-irritant compounds.

GLP compliance:

yes (incl. QA statement)

Details on study design:

MTT Direct Reduction Test
Prior to conducting the irritation assay, it was first necessary to determine if HCAT was capable of directly reducing MTT. Since the assay depends on the ability of viable cells to reduce MTT to formazan, any MTT reduction caused by the test item would interfere with the results. Four replicate samples (30 μL) of HCAT were added to a solution of MTT in PBS (300 μL, 0.5 mg/mL). Samples were then incubated for at least 3 h in an incubator set to maintain a temperature of 37°C. Production of reduced MTT (purple-coloured) was assessed by visual inspection. Four replicates of a known MTT reducing agent (eugenol, 30 μL) and the negative control (PBS, 30 μL) were tested in parallel to demonstrate the efficacy of the MTT solution.

Receipt of SkinEthic® HCE Units and Pre-incubation
After arrival at Charles River, the SkinEthic® HCE kit was inspected for quality. The pH and temperature indicators were both acceptable. Each individual unit was then transferred to separate wells of a 24 well plate containing maintenance medium (300 μL per well). Prior to dosing, all units were then incubated overnight in a humidified incubator set to maintain 37°C and 5% CO2.

Application of Test Item and Controls
Aliquots (30 μL) of HCAT were applied to seven replicate HCE units and 30 μL aliquots of the positive control (0.2% (w/v) Triton X-100) and the negative control (Dulbecco’s PBS) were applied to four replicate HCE units each using a positive displacement pipette. The pipette tip was used to gently spread the test item and controls over the entire exposed area of the SkinEthic® HCE units. The surface area of each HCE unit was 0.5 cm2, therefore the application rate was 60 μL/cm2.

Terminal Exposure Procedures (60 min Post Dose)
After exposure to the test item or positive and negative controls for 60 min, the HCE units were washed by rinsing with PBS (25 mL). The upper surface of the HCE unit was lightly swabbed with a cotton swab. After washing, all HCE units were blotted dry with tissue paper, transferred to fresh maintenance medium and incubated for ca 21 h in a humidified incubator set to maintain 37°C and 5% CO2.

MTT Test
After the recovery period, the relevant HCE units were transferred to a new 24 well plate containing MTT solution in SkinEthic maintenance medium (0.5 mg/mL, 300 μL per well). The three HCE units treated with HCAT to quantify the effect of any un-removed test item on the absorbance results were transferred to wells containing maintenance medium only (300 μL per well). Each unit was tapped gently on tissue paper to remove residual moisture before transferring to the MTT solution. The HCE units were then incubated for 3 h in an incubator set to maintain 37°C and 5% CO2.

Due to the physical nature of HCAT, it was not possible to completely remove the test item by washing. Cotton swabs were employed to facilitate the wash procedure but it was apparent that some test item remained on the surface of the HCE units. The effect of this un-removed test item on the absorbance values was calculated using additional HCE units dosed with HCAT. These additional units followed the same treatment as the other HCE units with the exception of the MTT incubation – this was replaced with a 3 h incubation in maintenance medium. The mean absorbance value of these was subtracted from the absorbance results for the HCE units dosed with HCAT and incubated with MTT.

Formazan Extraction
After incubation, each HCE unit was removed from the MTT solution or maintenance medium and gently tapped dry on tissue paper to remove any excess moisture. The HCE units were then transferred to wells containing 750 μL isopropanol to extract the formazan. A further 750 μL of isopropanol was also added to the upper surface of each HCE unit. The wells were then sealed and left for 90 min at room temp, protected from light, with gentle shaking.

Absorbance Measurement
Following the solvent extraction, the isopropanol from the upper and lower chambers was pooled and two aliquots (200 μL each) were then added to a 96 well plate for each sample. Plates were analysed using an MRX plate reader using wavelength measurement at 550 nm. Absorbance values were calculated against the background isopropanol sample contained on the plate.

Histology
After the recovery period, one HCE unit for each treatment was formalin for 3 h, then given a change of fixative before being fixed overnight. Following fixation, the HCE units were paraffin embedded and sections were stained by the standard haematoxalin-eosin method.

CALCULATIONS
Optical Density (OD)
OD Negative Control (NC) = OD NC raw – OD blank mean
The corrected mean OD Negative Control corresponds to 100% viability
OD Positive Control (PC) = OD PC raw – OD blank mean
OD Treated Tissue (TT) = OD TT raw – OD blank mean
OD Treated Tissue no MTT (TM) = OD TM raw – OD blank mean
OD Treated Tissue corrected (TC) = (OD TC raw – OD TM mean) – OD blank mean

Individual Viabilities (%)
% Positive Control 1 = [OD PC1 / Mean OD NC] x 100
% Positive Control 2 = [OD PC2 / Mean OD NC] x 100
% Positive Control 3 = [OD PC3 / Mean OD NC] x 100
% Treated Tissue 1 = [OD TT1 / Mean OD NC] x 100
% Treated Tissue 2 = [OD TT2 / Mean OD NC] x 100
% Treated Tissue 3 = [OD TT3 / Mean OD NC] x 100

Relative Viability (%)
% Viability = [ODTT / ODNC] x 100

Acceptance Criteria
The assay was deemed acceptable if the following criteria were met:
The mean OD value of the 3 negative control tissues was >0.6 and the Standard Deviation value (SD) of the % viability was <18.
The mean % viability of the 3 positive control tissues was <30% and the SD was <18.
The mean % viability SD of the 3 treated tissues was <18.

Interpretation of Eye Corrosion/Irritancy
The test chemical is considered to be a corrosive/irritant if the tissue viability after exposure and post-treatment incubation is less than 50% of the negative control.
Histology samples were also examined by light microscopy for signs of damage.

Irritation parameter:

other: Viability of HCE Unit

Basis:

other: PBS Solution (Negative Control)

Time point:

other: 16h

Max. score:

100

Reversibility:

not specified

Remarks:

N/A

Irritation parameter:

other: Viability of HCE Unit

Basis:

other: 0.2 % Triton X-100 (Positive Control)

Time point:

other: 16 h

Max. score:

6.67

Reversibility:

not specified

Remarks:

N/A

Irritation parameter:

other: Viability of HCE Unit

Basis:

other: HCAT

Time point:

other: 16 h

Max. score:

67.16

Reversibility:

not specified

Remarks:

N/A

Irritation parameter:

other: Viability of HCE Unit

Basis:

other: HCAT Tested without MTT

Time point:

other: 16 h

Max. score:

40.96

Reversibility:

not specified

Remarks:

N/A

Irritation parameter:

other: Viability of HCE Unit

Basis:

other: HCAT (Corrected)

Time point:

other: 16 h

Max. score:

26.19

Reversibility:

not specified

Remarks:

N/A

MTT Direct Reduction Test

The test was scored by visual assessment of the formation of purple-coloured formazan. The positive control (eugenol) reduced the MTT solution to purple-coloured formazan. The negative control (PBS) did not reduce MTT to formazan after 3 h incubation. Due to the deep red colour of HCAT, it was difficult to distinguish if MTT had been reduced. Therefore, three additional HCE units were dosed in parallel with the irritation assay to quantify the effect of any un-removed test item on the absorbance results.

Negative Control Groups

The negative control results were within the acceptance criteria defined in the ECVAM validation SOP and were therefore accepted.

Positive Control Groups

The positive control results were within the acceptance criteria defined in the ECVAM validation SOP and were therefore accepted.

HCAT

Viable HCE Units – Incubated Without MTT

The mean absorbance value of the three replicate HCE units dosed with HCAT and incubated without MTT was 0.339 ± 0.203. This value was subtracted from the absorbance value of the HCE units dosed with HCAT and incubated with MTT.

Viable HCE Units – Incubated With MTT

The results were similar for the three viable HCE units dosed with HCAT. Exposure to HCAT resulted in a mean HCE viability (corrected for effect of un-removed test item) of 26.19 ±16.03% of the negative control value.

Histology Results

There was no sign of tissue damage in the HCE units treated with PBS and HCAT. Tissue damage was observed in the HCE unit treated with Triton X-100.

Percentage Viability of HCE Units After at Least 16 h Recovery Time

Test Item	Replicate ID	Viability (%)	Mean	SD (%)	CV (%)
PBS Solution(Negative Control)	Rep 1, Rep 2, Rep 3	{96.11, 96.72}, {97.80, 96.84}, {106.87, 105.66}	100	4.90	4.90
0.2% Triton X-100 Solution (Positive Control)	Rep 1, Rep 2, Rep 3	{4.84, 4.96}, {3.39, 3.63}, {11.73, 11.48}	6.67	3.88	58.11
HCAT	Rep 1, Rep 2, Rep 3	{84.26, 81.97}, {48.36, 47.27}, {71.21, 69.88}	67.16	16.03	23.87
HCAT Tested without MTT	Rep 1, Rep 2, Rep 3	{26.84, 26.23}, {23.94, 23.45}, {73.26, 72.05}	40.96	24.59	60.02
HCAT(Corrected)	Rep 1, Rep 2, Rep 3	{43.30, 41.00}, {7.39, 6.31}, {30.24, 28.91}	26.19	16.03	61.19

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, HCAT was demonstrated to be an iirritant when tested in vitro using the SkinEthic® HCE reconstructed human corneal model.

Executive summary:

HCAT was tested in vitro using the SkinEthic® HCE reconstructed human corneal model. The endpoint of the assay was the estimation of cell viability by measuring the reduction of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to its formazan metabolite by mitochondrial reductase. Corrosive/irritant materials were identified by their ability to reduce cell viability below a threshold of 50% of the negative control value.

Prior to the conduct of the irritation assay, a preliminary test was conducted to assess the intrinsic ability of HCAT to reduce MTT to formazan. Due to the intense colour of the test item, three additional HCE units were dosed in parallel with the irritation assay to quantify the effect of any un-removed test item on the absorbance results.

The eye irritation potential was assessed by applying HCAT to the exposed surface of HCE reconstructed human corneal units application rate was 60 μL/cm2 for 60 min. After the exposure period, the test item was rinsed from the surface of the HCE unit using Dulbecco’s phosphate-buffered saline (PBS) (25 mL) and cotton swabs. The HCE units were then incubated for a recovery period (21 h) in a humidified incubator set to maintain temperature of 37°C and CO2 concentration of 5%.

Following the recovery period, HCE units destined for MTT cell viability were transferred to assay medium containing MTT (0.3 mg/mL). The “colour control” units were placed in fresh maintenance medium and returned to the incubator for 3 h. After incubation all HCE units (except those for histological assessment) underwent formazan extraction in isopropanol. The cell viability was assessed by measuring the optical density of the formazan at a wavelength of 550 nm.

Four replicates (three for MTT assay and one for histology) of the positive control, Triton X- 100 (30 μL, 0.2% w/v), and the negative control, PBS (30 μL) were tested in parallel to demonstrate the efficacy of the assay. The viability of each individual HCE unit was calculated as a percentage of the mean negative control viability (defined as 100%). The HCAT absorbance results were corrected to account for the effect of any test item not removed by the wash procedure.

Exposure to HCAT resulted in a mean HCE viability of 26.19 ±16.03% of the negative control value. Exposure to the positive control, Triton X-100 (0.2% w/v), resulted in a mean HCE viability of 6.67 ± 3.88% of the negative control value.

In conclusion, HCAT was demonstrated to be an irritant when tested in vitro using the SkinEthic® HCE reconstructed human corneal model.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No data are available for respiratory irritation.

Justification for selection of skin irritation / corrosion endpoint:
Only available study that meets the required quality criteria.

Justification for selection of eye irritation endpoint:
Only available study that meets the required quality criteria.

Effects on eye irritation: irritating

Justification for classification or non-classification

The SkinEthic® HCE reconstructed human corneal model is unable to define different categories of eye irritation for the purpose of classification and labelling. HCAT was therefore classified as Category 1 for eye irritation (H318: Causes serious eye damage).