Phosphoric acid, C8-10-alkyl esters

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 October 2016 to 07 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Confidential

IUPAC Name:

Confidential

Test material form:

liquid

Details on test material:

- Appearance/physical state: Amber liquid
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

other: adult

Justification for test system used:

INTRODUCTION
- The corrosivity potential of a chemical may be predicted by measurement of its cytotoxic effect, as reflected in the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] assay, on EPISKINTM (SM) reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD No. 404) and is specifically approved as a replacement for the in vivo skin corrosivity test within OECD No. 431.
- The present test is based on the experience that corrosive chemicals, formulations, products or mixtures show cytotoxic effects following short-term exposure of the stratum corneum of the epidermis. The purpose of this study is to predict the skin corrosivity potential of a chemical by assessment of its effect on a reconstituted human
epidermis.
- The EPISKINTM(SM) is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin corrosivity testing involves the topical application of test materials to the surface of the skin, and the subsequent assessment of their effects on cell viability. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT (Fentem et al., 1998). The test can be used for classification as not corrosive, or as 1A, 1B or 1C (COMMISSION REGULATION (EU) 2016/863 of 31 May 2016).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- Human skin: EPISKIN (SM) (Manufacturer: SkinEthic, France, Batch No.: 16-EKIN-040, Expiry Date: 10 October 2016) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
- Quality control: EPISKIN (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkin (SM) Test Kits used in the present study) and are documented in Appendix 2 (attached).
- Justification for selection of the test system: The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

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

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

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

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

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

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

PRE-INCUBATION (Day -1)
- The Maintenance Medium was pre-warmed to 37 °C.
- The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well).
- The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37 °C in an incubator with 5% CO2 in a > 95% humidified atmosphere.

APPLICATION (Day 0)
- The Assay Medium was pre-warmed to 37 °C.
- The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well).
- The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath.
- Two epidermis units were used for the test item or negative control or positive control substance in experiment.
- Test item (50 μL) was applied evenly to the epidermal surface of each of two test units.
- Physiological saline (50 μL) was added to each of the two negative control skin units.
- Glacial acetic acid (50 μL) was added to each of the two positive control skin units.
- The plates with the treated epidermis units were incubated for 4 hours (± 10 min) at room temperature (22.7 to 23.9 °C) covered with the plate lids.

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

MTT TEST (Day 0)
- MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two colour control units).
- The lid was replaced and the plate incubated at 37 °C in an incubator with 5% CO2 for 3 hours, protected from light.

FORMAZAN EXTRACTION (Day 0)
- At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were
placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
- The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (approximately 150 rpm) for formazan extraction.
- A blank sample containing 2 mL of acidified isopropanol was processed in parallel.
- Note: Before the epidermis was cut from each skin unit, white residue test item was found. However the remaining test item fell off after the epidermis was cut from each skin unit.

CELL VIABILITY MEASUREMENTS (Day 1)
- Following the formazan extraction, 2 × 200 μL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
- The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.

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

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

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

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

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

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

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

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

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

INTERPRETATION OF TEST RESULTS
- The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with OECD No. 431 (OECD, 2016). The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).
- For 2 disks: If both disks have mean viability of ≥ 35 % = Non Corrosive; if both disks have mean viability of < 35 % = Corrosive (at the corresponding incubation period).
- For more than 2 disks: If the mean value is ≥ 35 % and the variability is less than 50 % = Non Corrosive; if the mean value is < 35% and the variability is less than 50% = Corrosive.
- If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35 %. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL

Duration of treatment / exposure:

4 hours

Number of replicates:

Two

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

79

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ADDITIONAL CONTROLS
- As the test item was coloured, two additional test item-treated living tissues were used
for the non-specific OD evaluation.
- The mean optical density (measured at 570 nm) of these tissues was determined as 0.005.
- The Non Specific Colour % (NSC living %) was calculated as 0.6% (see Table 1, attached). This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

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

VALIDITY OF THE TEST
- After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions in each case.
- The mean OD value of the two negative control tissues was in the recommended range (0.974).
- The positive control treated tissues showed 0.7% viability demonstrating the proper performance of the assay.
- The difference of viability between the two test item-treated tissue samples in the MTT assay was 7.1%.
- The difference of viability between the two negative control tissue samples in the MTT assay was 4.8%.
- The mean OD value of the blank samples (acidified isopropanol) was 0.045.
- All these parameters were within acceptable limits and therefore the study was considered to be valid.
- Historical control data are presented in Appendix 3 (attached).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Mean cell viability was 79.0% compared to the negative control (after adjustment for non-specific MTT reduction) and the test item is considered to be non-corrosive to skin.

Executive summary:

GUIDELINE

An in vitro skin corrosivity test of test item was performed in a reconstructed human epidermis model. EPISKIN (SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay). The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

 

METHODS

Disks of EPISKIN (SM) (two units) were treated with test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSC living) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

 

RESULTS

 

Following exposure to test item, the mean cell viability was 79.0% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

 

CONCLUSION

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