Phosphoric acid, 2-ethylhexyl ester, ammonium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 January 2017 to 10 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidleines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium mono(2- ethylhexyl)phosphate

Molecular formula:

C8H22NO4P

IUPAC Name:

Ammonium mono(2- ethylhexyl)phosphate

Constituent 2

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:

C16H38NO4P

IUPAC Name:

Ammonium bis(2- ethylhexyl)phosphate

Constituent 3

Reference substance name:

2-ethylhexyl diphosphate ammonium salts / esters of 2-ethylhexan-1-ol with diphosphoric acid and their corresponding ammonium salts

Molecular formula:

C8H23NO7P2

IUPAC Name:

2-ethylhexyl diphosphate ammonium salts / esters of 2-ethylhexan-1-ol with diphosphoric acid and their corresponding ammonium salts

Test material form:

liquid

Details on test material:

Storage Conditions: Room temperature.

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated 2 - 8 ºC
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: In order to assess the potential non-specific reduction of the test article, 50 µL of test article was added to 1 mL of 1.0 mg/mL MTT and the colour change was assessed after incubation for 60 minutes at 37 ± 1 ºC, 5 ± 1% CO2, 95 % RH. There was no change in colour therefore the test article was deemed not to interact with MTT.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Warmed to 37 ºC to enable easier pipetting of the viscous test item.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as supplied - viscous liquid.

OTHER SPECIFICS: n/a

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed epidermis with a functional stratum corneum supplied by MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM Reconstructed Human Epidermis
- Tissue batch number(s): 00267; EpiDerm RHE kit lot #: 23338
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: Not reported
- Date of initiation of testing: 18 January 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Not reported
- Temperature of post-treatment incubation (if applicable): 37 ± 1 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsed with PBS, volume not reported.
- Observable damage in the tissue due to washing: Not reported, assume no.
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Yes, details not reported
- Wavelength: 570 nm
- Filter: No / not reported
- Filter bandwidth: No / not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: acceptable (reported in CoA)
- Barrier function: acceptable (reported in CoA)
- Morphology: acceptable (reported in CoA)
- Contamination: acceptable (reported in CoA)
- Reproducibility: COV of mean replicated was <30 %.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Freeze killed tissues
- Procedure used to prepare the killed tissues (if applicable): Not reported
- No. of replicates : 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: n/a

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): n/a

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

incubated in MTT medium for 3 hours and followed by an overnight extraction in isopropanol.

Number of replicates:

2 for each timepoint

Test animals

Species:

other: n/a

Strain:

other: n/a

Details on test animals or test system and environmental conditions:

n/a

Test system

Type of coverage:

other: n/a

Preparation of test site:

other: n/a

Vehicle:

other: n/a

Amount / concentration applied:

n/a

Duration of treatment / exposure:

n/a

Observation period:

n/a

Number of animals:

n/a

Details on study design:

n/a

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 repeat - 3 min exposure

Value:

79

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100 % viability (OD 1.221)

Positive controls validity:

valid

Remarks:

4 % viability

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 repeat - 60 min exposure

Value:

17.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100 % viability (OD 1.394)

Positive controls validity:

valid

Remarks:

0 % viability

Remarks on result:

no indication of irritation

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: Yes

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
- Range of historical values if different from the ones specified in the test guideline: n/a

Any other information on results incl. tables

Table 2:      Mean OD₅₇₀Values and Viabilitiesfor the Negative Control Item, Positive Control Item and Test Item (Repeat Experiment 1)

Tissue	Exposure Period	MeanOD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.190	1.221	0.040	3.2	100*
		1.252
	60 Minutes	1.532	1.394	nd	10.9
		1.256
Positive Control	3 Minutes	0.296	0.301	0.028	8.0	4.0
		0.305
	60 Minutes	0.127	0.128	nd	4.9	-0.8
		0.130
Test Item	3 Minutes	1.140	0.962	0.196	20.4	79
		0.783
	60 Minutes	0.277	0.243	nd	15.6	17.5
		0.210

OD = Optical density

*=  The mean % viability of the negative control tissue is set at 100 %

nd= Not determined

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study the test substance is not considered to be corrosive in the in vitro skin corrosion test using the EpiDerm Skin Model.

Executive summary:

OECD 431 (2017) - The skin corrosivity potential of reaction mass of ammonium mono(2 ethylhexyl)phosphate, ammonium bis(2 ethyl hexyl)phosphate and 2 -ethylhexyl diphosphate ammonium salts was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 79 % and 17.5 %, respectively. The quality criteria required for acceptance of the resuts was met.

Under the conditions of this study the test substance is not considered to be corrosive to the skin.