[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-04-05 to 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium bis(2-ethylhexyl) phosphate

EC Number:

225-615-3

EC Name:

Ammonium bis(2-ethylhexyl) phosphate

Cas Number:

4971-47-5

Molecular formula:

C16H35O4P.H3N

IUPAC Name:

ammonium bis(2-ethylhexyl) phosphate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

The Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SCT) was used as test system (MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic).

Justification for test system used:

As recommended in OECD Guideline No. 431, Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SCT) has been selected as test system for in vitro skin irritation. The RhE test system uses human derived non-transformed keratinocytes as cell source to reconstruct an epidermal model with representative histology and cytoarchitecture.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SCT)
- Tissue batch number: 25892

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 15 times rinsing with sterile DPBS , the constant stream of DPBS was applied from the nearest distance from the tissue surface. After the 15th rinse with washing bottle, the inserts were completely submerged 3 times in approximately 50 mL of DPBS and shaken to remove all traces of test item/control item. Finally, each tissue was rinsed once from inside and once from outside with sterile DPBS. The excess of DPBS was removed by gently shaking the insert and blotting the insert on sterile blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 2 hours and 55 minutes
- Spectrophotometer: plate reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
None - The test substance did not directly reduce MTT.

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered corrosive according to UN GHS (Category 1) if the mean tissue viability after 3 minutes exposure is < 50% or
- The tissue viability after 3 minutes exposure is ≥50% and <15% after 1 hour exposure.

The test item is considered as non-corrosive to skin in accordance with UN GH, if the tissue viability after 3 minutes exposure is ≥50% and ≥15% after 1 hour incubation exposure.

Step 2
The test item identified as being corrosive in step 1 is further subcategorised in accordance with UN GHS based on the following:
The tissue viability after 3 minutes exposure is <25%: optional subcategory 1A.
The tissue viability after 3 minutes exposure is ≥25 %: a combination of optional Sub-Categories 1B-and-1C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 minutes and 60±1 minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

Test item, positive control and negative control were tested in duplicates.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

test item

Run / experiment:

1 - 3 min exposure

Value:

35.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2 - 60 min exposure

Value:

4.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Remarks:

negative control

Run / experiment:

1 - 3 min exposure

Value:

100

Vehicle controls validity:

not examined

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

% tissue viability

Remarks:

negative control

Run / experiment:

2 - 60 min epxosure

Value:

100

Vehicle controls validity:

not examined

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 5

Irritation / corrosion parameter:

% tissue viability

Remarks:

positive control

Run / experiment:

1 - 3 min exposure

Value:

6.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not applicable

Results 6

Irritation / corrosion parameter:

% tissue viability

Remarks:

positive control

Run / experiment:

2 - 60 min exposure

Value:

6.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not applicable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The technical proficiency of the test method was established by using proficiency chemicals under Bioneeds Study No.: BIO-GT 1000, according to OECD Test Guideline No. 431.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

TABLE 1.           Summary of optical density (OD) and viability (%)

 

3 Minutes Exposure                                                                                        Refer Appendix - 1

Treatment		OD	Viability (%)	Classification
Negative Control (Sterile water)	Mean	1.609	100.0	NC
	±SD	0.030	2.6
	n	2	2
Positive Control (Glacial acetic acid)	Mean	0.100	6.2	C (Category 1A)
	±SD	0.004	0.4
	n	2	2
Test Item [Phosphoric acid, 2-etylhexyl ester, NH4+ salt (Primasol NF)]	Mean	0.566	35.2	C (Category 1A)
	±SD	0.031	2.7
	n	2	2

                                           

 

1 Hour Exposure

Treatment		OD	Viability (%)	Classification
Negative Control (Sterile water)	Mean	1.555	100.00	NC
	±SD	0.034	3.1
	n	2	2
Positive Control (Glacial acetic acid)	Mean	0.104	6.7	C (Category 1A)
	±SD	0.003	0.3
	n	2	2
Test Item [Phosphoric acid, 2-etylhexyl ester, NH4+ salt (Primasol NF)]	Mean	0.070	4.5	C (Category 1A)
	±SD	0.005	0.4
	n	2	2

NC = Non Corrosive; C = Corrosive; n = No. of tissues; SD = Standard Deviation.

    

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Remarks:

The combination of sub-categories 1B and 1C applies

Conclusions:

Based on the results obtained under the conditions of this study according to OECD 431 guideline, the test item is considered as corrosive to skin in accordance with UN GHS (sub-categories 1B and 1C), as the mean percentage tissue viability was less than 50% but >= 25% of the negative control after 3 minutes exposure.

Executive summary:

The objective of this study was to evaluate in vitro skin corrosion potential of the test item by measurement of tissue viability on the Epidermal Model - Epiderm™ (EPI-200-SCT) as per the OECD Guideline for the testing of chemicals No. 431, “In vitro skin corrosion: reconstructed human epidermis (RHE) test method”, adopted on 29th July 2016. The test item did not develop any colour when dissolved in distilled water/isopropanol and is considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. Tissues were visually inspected for any defects such as air bubble or excess moisture observed all the tissue inserts were used for the study. Tissue inserts were transferred to upper row of 6 well plates prefilled with 0.9 mL of assay medium and incubated in CO2 incubator for 60 minutes.

Test item were exposed for 1 hour and 3 minutes separately. All the treatments were maintained in duplicates. For 3 minutes treatment, quantity of 50 µL of sterile distilled water (NC) was dispensed into the first insert atop the tissue. After 60 seconds the procedure was repeated with second tissue and continued for other tissues. Similar procedure was followed in the same manner until all the tissues were treated. Tissues were treated with 50 µL of test item and 50 µL of positive control (glacial acetic acid). For 1 hour treatment, quantity 50 µL of test item, 50 µL of negative control and 50 µL of positive control were dispensed directly atop Epiderm™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 1 hour. At the end of treatment time tissue inserts were rinsed with sterile PBS (fill and empty insert 20 times in a constant soft stream of 1xPBS) to remove any residual test item. Post rinsing procedure, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and incubated for 3 hours at 37±1°C and 5±1% CO2. Post incubation, the tissue inserts were removed and blotted onto the tissue paper and transferred to a pre-labeled 24-well plate containing 2.0 mL of isopropanol in each designated. The plates were placed on an orbital plate shaker and shaken (̴ 120 rpm/minute) for 4 hours and 15 minutes (for 1 hour exposure) and 4 hours and 49 minutes (for 3 minutes exposure) at room temperature. At the end of the extraction period, the tissue was pierced with an injection needle and allowed the extract to run into the well from which the insert was taken. The punctured inserts were discarded and solution was placed on mixer for 15 minutes until it became homogenous. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated. For 3 minutes exposure, percentage viability of negative control, positive control and test item was 100±2.6, 6.1±0.4 and 35.1±2.7, respectively. For 1 hour exposure, percentage viability of negative control, positive control and test item was 100±3.1, 6.7±0.3 and 4.5±0.4 respectively.

According to the prediction scheme shown in Table 5 of OECD 431 (version dated 18 June 2019) the test item is predicted to be corrosive and placed in the combination of sub-categories 1B and 1C as the viability after 3 min exposure is < 50% but >=25% (step 1 and 2 of the prediction scheme, respectively).