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

Skin irritation / corrosion

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

700-831-5

Molecular formula:

Mo2O2L8 (L = 2-ethylhexanoic acid; C8H16O2) (L' = 2-ethylhexanoic acid; C8H16O2)

IUPAC Name:

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

Test system

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.

Results and discussion

In vitro

Results

Results 1

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)

Results 2

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)

Results 3

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)

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.