2-Propenoic acid, 2-hydroxyethyl ester, reaction products with O,O-bis(dialkyalkyl) hydrogen phosphorodithioate and dimethyl phosphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 July 2019 to 04 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B40 bis (In Vitro Skin Corrosion Human Skin Model Test)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Details on test material:

- Appearance/physical state: Light yellow liquid
- Storage conditions: Ambient temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

other: neonatal

Vehicle:

unchanged (no vehicle)

Details on test system:

PURPOSE OF THE TEST
- The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes.
- Corrosion is directly related to cytotoxicity in the EpiDerm tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.
- This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS AND MTT
- The negative control item was used as supplied.
- The positive control item was used as supplied.
- A 1.0 mg/mL MTT solution was prepared from a MatTek MTT-100 kit immediately prior to use.

EPIDERM RECONSTRUCTED HUMAN EPIDERMIS MODEL KIT
- Supplier: MatTek In Vitro Life Sciences Laboratories
- Date received: 02 July 2019
- EpiDerm tissues (0.63 cm2) lot number: 30804
- Assay medium lot number: 062719MSC
- Upon receipt of the Epiderm tissues, the sealed 24-well plate was stored in a refrigerator until use.

MTT DYE METABOLISM, CELL VIABILITY ASSAY
- The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells.
- One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

TEST FOR DIRECT MTT REDUCTION
- A test item may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
- Test item (50 µL) was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
- If the MTT solution containing the test item turned blue/purple relative to the control, the test item was presumed to have reduced the MTT.
- The MTT solution containing the test item turned a brown color as opposed to blue/purple. Therefore, even though the solution did not blue/purple, the darkening to brown indicated that the test item may have the potential to directly reduce MTT and corrective procedures should be undertaken to avoid a false negative result. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and freeze-killed tissues.
- This step was a functional check which employs freeze-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues.
Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERM tissues in an empty 12-well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
- In addition to the normal test procedure, the MTT reducing test item was applied to two freeze-killed tissues per exposure period. In addition, two freeze-killed tissues per exposure period remained untreated. The untreated freeze-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- A test item may interfere with the MTT endpoint if it is colored or if it becomes colored when in wet or aqueous conditions. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
- Test item (50 μL) was added to 300 μL of sterile water. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. A visual assessment of the colour was then made.

MAIN TEST PRE-INCUBATION
- The assay medium was brought to room temperature before use.
- An aliquot (0.9 mL) of this assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3-minute and 60-minute exposure periods.
- EpiDerm tissues were transferred into the 6-well plates containing the assay medium.
- The 6-well plates containing the EpiDerm samples were pre-incubated (37 °C, 5 % CO2) for approximately 1 hour before dosing.

APPLICATION OF TEST ITEM AND RINSING
- Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-minute and 60-minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT-loading. Another 24-well plate was prepared for the MTT-loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT-loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
- After pre-incubation of the EpiDerm tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure. For the 60-Minute exposure period, 50 μL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 μL of the test item and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period.
- When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT-loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated.
- After the 3-hour MTT incubation was complete, the tissue inserts were blotted and transferred to 24-well plates for formazan (reduced MTT) extraction. The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol. The plate was covered with plate sealer, to prevent isopropanol evaporation, and stood overnight at room temperature, to allow extraction to proceed.

ABSORBANCE / OPTICAL DENSITY MEASUREMENTS
- After extraction, each tissue was pierced with a pipette fitted with a 1000 μL tip and the extraction solution was forced vigorously up and down to form a homogenous solution.
- Aliquots (3 x 200 µL) of the extract were transferred to the appropriate wells of a pre-labelled 96-well plate.
- Isopropanol (200 µL) alone was added to the three wells designated as blanks.
- Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader and LT-com analysis software.
- Servicing, calibration, pass band width and linearity range of the microplate reader is given in Annex 1 (attached).

QUANTITATIVE MTT ASSESSMENT (PERCENTAGE TISSUE VIABILITY)
- The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water.
- The relative mean viabilities were calculated using the equation relative mean viability (%) = (mean OD570 of test item / mean OD570 of negative control) x 100.
- It was determined that the test item may have the potential to directly reduce MTT and therefore freeze-killed tissues were employed, the results of the MTT assay were corrected using the equation true viability = mean OD tvt – (OD tkt – OD ukt) where OD = optical density at 570 nm; tvt = treated viable tissues, tkt = treated killed tissues; ukt = untreated killed tissues.
- If direct reduction by the test item is greater than 30 % of the negative control value, additional steps must be taken into account or the test item may be considered incompatible with this test system.
- If direct reduction by the test item is less than 30 % of the negative control value, the mean OD of the test item treated killed control may be subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

QUALITY CRITERIA
- The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
(i) Negative control: The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
(ii) Positive control: Potassium hydroxide 8.0 N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60-minute positive control is < 15%.
(iii) Coefficient of variation: In the range 20 and 100 % viability, the Coefficient of Variation between tissue replicates should be ≤ 30 %.

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System.
- Labtech LT-4500 microplate reader and LT-com analysis software.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL of test item

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Duplicate tissues

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

93.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean relative viability (% of negative control)

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

6-minute exposure

Value:

47

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean relative viability (% of negative control)

Other effects / acceptance of results:

DIRECT MTT REDUCTION
- The MTT solution containing the test item turned a brown colour as opposed to blue/purple. Therefore, even though the solution did not blue/purple, the darkening to brown indicated that corrective procedures should be undertaken to avoid a false negative result. Therefore, an additional procedure using freeze-killed tissues was performed.
- The results of the freeze-killed tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

INTERFERENCE BY THE TEST ITEM RELATIVE TO THE CORRESPONDING NEGATIVE CONTROL
- 3 minutes exposure: Mean of test item treated killed tissues (tkt) = 0.144 OD570; Mean of untreated killed tissues (ukt) = 0.148 OD570. The direct reduction by the test item relative to the negative control value: (0.144 (tkt) – 0.148 (ukt)) / 2.269 (mean of negative control) = 0.0% (negative value treated as 0.0% to avoid artificially inflating the results).
- 60 minutes exposure: Mean of test item treated killed tissues (tkt) = 0.223 OD570; Mean of untreated killed tissues (ukt) = 0.135 OD570. The direct reduction by the test item relative to the negative control value: (0.223 (tkt) – 0.135 (ukt)) / 2.362 (mean of negative control) = 3.7 %.
- The interference by the test item relative to the corresponding negative control was 0.0 % after 3 minutes exposure and 3.7 % after 60 minutes exposure. Therefore, direct reduction was < 30 % relative to the negative control and considered acceptable.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test item did not become coloured.
- This observation was taken to indicate the test item did not have the potential to cause colour interference.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- Mean OD570 values and viabilities for the negative control, positive control and test item are given in Appendix 1 (attached).
- The relative mean viabilities for each treatment group are shown in the table below.

QUALITY CRITERIA
- The mean OD570 for the negative control treated tissues was 2.269 for the 3-minute exposure period and 2.362 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- The relative mean tissue viability for the positive control treated tissues was 2.5 % relative to the negative control following the 60-minute exposure period. The positive control acceptance criterion was therefore satisfied.
- In the range 20 to 100 % viability, the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30 %. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

RELATIVE MEAN VIABILITIES FOR EACH TREATMENT GROUP

Exposure period	Percentage viability negative control*	Percentage viability positive control	Percentage viability test item
3 minute	100	3.3	93.6
60 minute	100	2.5	47.0

* Mean viability of the negative control tissues is setat 100%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean viability of the test item treated tissues was 93.6 % after 3 minutes exposure and 47.0 % after 60 minutes exposure.

Executive summary:

GUIDELINE

The study was performed in compliance with the OECD Guideline for the Testing of Chemicals No 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (29 July 2016) and Method B.40bis of Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

METHODS

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. It was determined that the test item may have the potential to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). Data were presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

RESULTS

Mean viability of the negative control tissues was set at 100 % and quality criteria for acceptance of results were satisfied. Relative mean viabilities after 3 minutes exposure were determined to be 100 % (negative control), 3.3 % (positive control) and 93.6 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 2.5 % (positive control) and 47.0 % (test item).

 

CONCLUSION

The relative mean viability of the test item treated tissues was 93.6 % after 3 minutes exposure and 47.0 % after 60 minutes exposure.