Phosphoric acid, 2-ethylhexyl ester, ammonium salt

Endpoint summary

Description of key information

Skin Irritation/Corrosion

Not corrosive; OECD 431; Anon., 2017

Skin irritant (Skin Irrit. 2); OECD 439; Anon., 2017

Eye Irritation/Corrosion

Inconclusive; OECD 437; Anon., 2017

Eye Irritant (Eye Irrit. 2); OECD 492; Anon., 2018

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

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

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

Species:

other: n/a

Strain:

other: n/a

Details on test animals or test system and environmental conditions:

n/a

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

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

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

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

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 2017 to 08 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not reported

Source strain:

other: n/a

Details on animal used as source of test system:

n/a

Justification for test system used:

n/a

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Reconstructed Human Epidermis, supplied by MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic
- Tissue batch number(s): 00267; EpiDerm kit lot # = 25801
- Production date: not reported
- Shipping date: not reported
- Delivery date: not reported
- Date of initiation of testing: 22 March 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 ºC
- Temperature of post-treatment incubation (if applicable): 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsed with PBS (volume and number of rinsings not reported)
- Observable damage in the tissue due to washing: 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: not reported
- Wavelength: 570 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: acceptable (provided in CoA)
- Barrier function: acceptable (provided in CoA)
- Morphology: acceptable (provided in CoA)
- Contamination: acceptable (provided in CoA)
- Reproducibility: The standard deviation (SD) for tissues should be ≤18 %.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- n/a

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after exposure is less than 50 %, realtive to the negative control.
- The test substance is considered to be non-irritant to skin if the viability after exposure is greater than or equal to 50 %, relative to the negative control.
- 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): 30 µL
- Concentration (if solution): undiluted

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): 30 µL
- Concentration (if solution): not reported

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % w/v

Duration of treatment / exposure:

60 mins

Duration of post-treatment incubation (if applicable):

Post exposure, the tissues were placed on the appropriate medium and incubated for 41 hours and 35 minutes.

Upon completion of the recovery period, each tissue was rinsed with PBS before being placed on 0.3 mL of 1 mg/mL MTT in PBS and incubated for three hours at 37 ºC.

Number of replicates:

3

Species:

other: n/a

Strain:

other: n/a

Details on test animals or test system and environmental conditions:

n/a

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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three replicates

Value:

2.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive 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: The OD values for the negative controls were between 0.8 and 2.8.
- Acceptance criteria met for positive control: Viability of tissues was ≤20 % (3.7 %).
- Acceptance criteria met for variability between replicate measurements: Yes, ≤18 % (ca. 4 % for both negative and positive control groups).
- Range of historical values if different from the ones specified in the test guideline: n/a

Table 2:      Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)	% COV Relative mean viability (%)
Negative Control Item	1.136	104.3	100*	4.2	4.2
	1.044	95.9
	1.086	99.8
Positive Control Item	0.039	3.5	3.7	0.1	3.6
	0.041	3.8
	0.041	3.7
Test Item	0.024	2.2	2.5	0.7	28.4
	0.022	2.0
	0.037	3.4

OD= Optical Density

SD= Standard deviation

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

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of this study the test substance is considered to be irritant to the skin and can be preliminarily classfied as Category 2 in accordance with UN GHS and EU CLP Regulation.

Executive summary:

OECD 439 (2017) - The skin irritation potential of the test item was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP.

Triplicate tissues were exposed to the test item for 60 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT. After incubation and extraction, the solutions were 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 60 minutes were 2.5 %. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The quality criteria required for acceptance of the results was met.

Under the conditions of this study the test substance is considered to be irritant to the skin and can be preliminarily classfied as Category 2 in accordance with UN GHS and EU CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 2017 to 08 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guideliens and in accordance with GLP. All guidelines validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir.
- Number of animals: not reported
- Characteristics of donor animals (e.g. age, sex, weight): not reported
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):The eyes were removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) in a suitably sized container and transported on the same day to the testing facility.
- Time interval prior to initiating testing: same day
- indication of any existing defects or lesions in ocular tissue samples: only corneas free of defects were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL solution used to transport eyes to testing facility.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 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

Duration of treatment / exposure:

10 mins

Duration of post- treatment incubation (in vitro):

Following exposure and rinsing, corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour and 25 minutes at 32±1°C.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

QUALITY CHECK OF THE ISOLATED CORNEAS: The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

NUMBER OF REPLICATES: 3 per treatment group.

NEGATIVE CONTROL USED: The negative control substance was 0.9% sodium chloride solution supplied by Baxter Healthcare Ltd., Thetford, UK.

SOLVENT CONTROL USED (if applicable): n/a

POSITIVE CONTROL USED: The positive control substance was dimethylformamide supplied by Sigma-Aldrich Company Ltd., Poole, UK.

APPLICATION DOSE AND EXPOSURE TIME: 10 mins

TREATMENT METHOD: Not reported

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After exposure, each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). Cotton buds were used to aid the removal of the test article. The corneas were then washed once in media without phenol red.

- POST-EXPOSURE INCUBATION: After test item exposure and rinsing, the corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour and 25 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opaciometer.
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of spectrophotometry (OD 490).
- Others (e.g, pertinent visual observations, histopathology): (please specify) n/a

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. As per OECD 437.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean value

Value:

33.93

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control yields opacity and permeability values that are less than the established upper limits for these endpoints for bovine corneas as treated at the testing facility.
- Acceptance criteria met for positive control: The positive control yields an IVIS within 2 standard deviations of the historical control mean.
- Range of historical values if different from the ones specified in the test guideline: n/a

Table 1       Corneal Opacity

Test Substance	Cornea Number	Initial Opacity	Post Incubation Opacity	Change in Opacity	Mean Change in Opacity	Corrected Opacity	Mean Corrected Opacity
Test article	7	-2	9	11	N/A	11.0	12.7
	8	-2	11	13		13.0
	15	-2	12	14		14.0
Negative	1	-2	-2	0	0.00	0.0	0.0
	2	-2	-2	0		0.0
	6	-2	-2	0		0.0
Positive	3	-3	66	69	N/A	69.0	73.3
	16	-2	72	74		74.0
	17	-2	75	77		77.0

 

Table 2       Corneal Permeability

Test Substance	Cornea Number	Mean Blank OD490	OD490	Corrected OD490	Mean Corrected OD490	Final Corrected OD490	Mean Group Corrected OD490
Test article	7		1.435	1.435	N/A	1.426	1.417
	8		1.444	1.444		1.435
	15		1.400	1.400		1.391
Negative	1	0.000	0.009	0.009	0.009	0.000	0.000
	2		0.005	0.005		-0.004
	6		0.014	0.014		0.004
Positive	3		0.274	0.274	N/A	0.265	0.421
	16		0.308	0.308		0.299
	17		0.708	0.708		0.698

 

Table 3       Calculated IVIS

Test Chemical	Mean Opacity	Mean Permeability	IVIS (Mean Opacity + (15 x Mean Permeability))
Test article	12.7	1.417	33.93
Negative control	0.00	0.000	0.000
Positive control	73.3	0.421	79.64

Interpretation of results:

study cannot be used for classification

Conclusions:

It was concluded that under the condition of this study, the test item, produced an IVIS score of 33.93 and therefore no prediction could be made in respect of its potential to cause eye irritation.

Executive summary:

OECD 437 (2017) - The Bovine Corneal Opacity and Permeabilty (BCOP) test was conducted using reaction mass of the test item in accordance with OECD Guideline 437 "Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage" (2013). Undiluted test item was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared. Measurements for corneal opacity were made after 2 h incubation in the horizontal position with fresh media. Measurements for corneal permeability were made following 1 h and 25 min incubation in the vertical position with sodium fluorescein. Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD₄₉₀). The mean corrected opacity reading and permeability readings for the test item were 12.7 and 1.417, resulting in an In Vitro Irritation Score (IVIS) of 33.93. As a result, no prediction could be made in respect to the test items potential to cause eye irritation.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July 2017 to 03 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guideliens and in accordance with GLP. All guidelines validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ).
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (29 November 2016) of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.

Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (about 17 hours).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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

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

Duration of treatment / exposure:

30 mins

Duration of post- treatment incubation (in vitro):

120 mins

Number of animals or in vitro replicates:

Each group tested in duplicate

Details on study design:

- Details of the test procedure used

Pre Test Assessments

The test items ability to directly reduce MTT was assessed. For this purpose approximately 50 µl of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. The result indicated that the test item did not have MTT reducing effects. An additional test with freeze-killed tissues did not have to be performed.

The photometric properties of the test item after contact with water and isopropanol were assessed. For this purpose each 50 µL of the test item were added to 1.0 ml of water and to 2 ml isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for at least one hour, the isopropanol mixture or for 2 to 3 hours at room temperature. The result indicated that the test item did not dye the water or isopropanol and would therefore not interfere with the photometric assessment of MTT during the main test.

Main Test

After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes.

After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 µL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.

At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilised a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).

After rinsing, the tissues were immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.

At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 ml of warm Assay Medium. The tissues are incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

After post-treatment incubation of 120 minutes the MTT assay was performed.

At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 ml of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.

The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

- RhCE tissue construct used, including batch number: EpiOcular kit (Lot: 27002). Supplied by MatTek Coorp., Slovakia.

- Doses of test chemical and control substances used: 50 µL applied topically.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Tissues exposed for 30 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH. Tissues were immersed for 2 seconds during the rinsing process. Post exposue incubation was 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2.

- Justification for the use of a different negative control than ultrapure H2O (if applicable): N/A

- Justification for the use of a different positive control than neat methyl acetate (if applicable): N/A

- Description of any modifications to the test procedure: N/A

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): N/A

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per test group (test item, positive and negative control). 1 blank tissue prepared with no treatment.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm

- Description of the method used to quantify MTT formazan: See above.

- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): N/A

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The percent viability of each of the two relating tissues for each control and test item relative to the negative control (100% control) were calculated. The difference of the viability between duplicate tissues was calculated. If the difference is >20% the test is considered as non-qualified. The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model. If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes

- Complete supporting information for the specific RhCE tissue construct used: Yes

- Reference to historical data of the RhCE tissue construct: Yes

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Yes

- Positive and negative control means and acceptance ranges based on historical data: Yes

- Acceptable variability between tissue replicates for positive and negative controls: Yes

- Acceptable variability between tissue replicates for the test chemical: Yes

Irritation parameter:

other: Spectrophotometric mean relative absorbance of MTT solution at 570 nm

Run / experiment:

Mean value

Value:

1.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- The negative control OD is > 0.8 and < 2.5 (1.674 and 1.753).
- The mean relative viability of the positive control is below 50% of the negative control viability (36.5%).
- The difference of viability between the two relating tissues of a single item is < 20% (values between 0.6% and 1.8%) in the same run (for positive and negative control tissues and tissues of single test items).

Table 1. Results after exposure to Reaction mass of Ammonium mono(2-ethylhexyl)phosphate, Ammonium bis(2-ethylhexyl)phosphate and 2-ethylhexyl diphosphate ammonium salts and the controls

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Mean OD of Treatment Group blank corrected	Rel. Viability [%] Tissue 1, 2	Absolute Value of the Difference of Rel. Viability  Tissue 1,2[%]	Mean Rel. Viability [%]**
Blank		0.038	0.038	0.038
Negative Control	1	1.674	1.753	1.714	1.676	1.691	99.1	1.8	100.0
	2	1.735	1.753	1.744	1.706		100.9
Positive Control	1	0.626	0.693	0.660	0.622	0.617	36.8	0.6	36.5
	2	0.647	0.652	0.649	0.611		36.2
Test Item	0.078	0.083	0.081	0.043		0.033	2.5	1.2	1.9
	0.061	0.060	0.061	0.023			1.3

* mean of 2 replicates after blank correction

** relative absorbance = ((100 • test item absorbance) / mean absorbance of the negative control)

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

It can be stated that in this study and under the experimental conditions reported, the test item possesses eye irritating potential.

Executive summary:

OECD 492 (2018) - This in vitro study was performed to assess the eye irritation potential of Reaction mass of the test item by means of the Human Cornea Model Test. Additional tests with viable or freeze-killed tissues were not performed, since the colourless test item did not dye water or isopropanol, and did not prove to be a MTT reducer. Tissues of the human cornea model EpiOcular™were treated with the test item, the positive and the negative control for 30 minutes each in duplicate. 50 µL of the test item and of the controls, respectively, were applied to each tissue, spread to match the tissue size. Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 36.5 %, thus the validity of the test system is ensured. Relevant irritating effects were observed following 30 minutes incubation with the test item. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 1.9 % compared with the value of the negative control (threshold for irritancy: ≤ 60%). In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses an eye irritating potential, category 2 according to the CLP Regulation and UN GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion

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 30 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.

Skin Irritation

OECD 439 (2017) -The skin irritation potential of reaction mass of ammonium mono (2 -ethylhexyl)phosphate, ammonium bis(2 -ethylhexyl)phosphate and 2 -ethylhexyl diphosphate ammonium salts was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP.

Triplicate tissues were exposed to the test item for 60 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT. After incubation and extraction, the solutions were 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 60 minutes were 2.5 %. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The quality criteria required for acceptance of the results was met.Under the conditions of this study the test substance is considered to be irritant to the skin and can be preliminarily classfied as Category 2 in accordance with UN GHS and EU CLP Regulation.

Eye Irritation/Corrosion

OECD 437 (2017) - The Bovine Corneal Opacity and Permeabilty (BCOP) test was conducted using reaction mass of ammonium mono(2-ethylhexyl)phosphate, ammonium bis(2-ethylhexyl)phosphate and 2-ethylhexyl diphosphate ammonium salts in accordance with OECD Guideline 437 "Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage" (2013).

Undiluted test item was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared. Measurements for corneal opacity were made after 2 h incubation in the horizontal position with fresh media. Measurements for corneal permeability were made following 1 h and 25 min incubation in the vertical position with sodium fluorescein. Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD₄₉0). The mean corrected opacity reading and permeability readings for the test item were 12.7 and 1.417, resulting in an In Vitro Irritation Score (IVIS) of 33.93.As a result, no prediction could be made in respect to the test items potential to cause eye irritation.

Eye Irritation

OECD 492 (2018) - This in vitro study was performed to assess the eye irritation potential of Reaction mass of Ammonium mono(2-ethylhexyl)phosphate, Ammonium bis(2-ethylhexyl)phosphate and 2-ethylhexyl diphosphate ammonium salts by means of the Human Cornea Model Test. Additional tests with viable or freeze-killed tissues were not performed, since the colourless test item did not dye water or isopropanol, and did not prove to be a MTT reducer. Tissues of the human cornea model EpiOcular™were treated with the test item, the positive and the negative control for 30 minutes each in duplicate. 50 µL of the test item and of the controls, respectively, were applied to each tissue, spread to match the tissue size. Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 36.5 %, thus the validity of the test system is ensured. Relevant irritating effects were observed following 30 minutes incubation with the test item. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 1.9 % compared with the value of the negative control (threshold for irritancy: ≤ 60%). In conclusion, it can be stated that in this study and under the experimental conditions reported, Reaction mass of Ammonium mono(2-ethylhexyl)phosphate, Ammonium bis(2-ethylhexyl)phosphate and 2-ethylhexyl diphosphate ammonium salts possess an eye irritating potential, category 2 according to the EU CLP Regulation and UN GHS.

Justification for classification or non-classification

The substance meets the criteria for the following classifications in accordance with Regulation (EC) No 1272/2008 (CLP):

- Skin Irrit. 2 ( H315: Causes skin irritation)

- Eye Irrit. 2 ( H319: Causes serious eye irritation)