Phosphoric acid, 2-ethylhexyl ester, sodium salt

Endpoint summary

Description of key information

Based on results of in vitro studies on skin irritation/corrosion according to OECD 439 and 431, respectively, the test item was considered to be irritating to skin. However, due to the results observed in the acute dermal toxicity study in rats which had to be terminated after two days due to animal welfare reasons as corrosive signs have been found on the skin surface of the treated animal (please refer to IUCLID section 7.2.3), the substance is considered to be classfied as corrosive to skin (Cat 1B), taking into account the 24 hours exposure period in this study.

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test, according to OECD 437, the test item, induced an IVIS of 70.7 at 10 minutes of treatment. As the test item induced an IVIS >55 it is considered as severe irritant/causing serious eye damage to bovine cornea and classified as UN GHS category 1.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-03-22 to 2018-03-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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-SIT) 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. 439, Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SIT) 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-SIT)
- Tissue batch number: 25888

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 identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean tissue viability after exposure and post-treatment incubation is ≤50%.
- The test substance is considered as non-irritant to skin in accordance with UN GHS No Category, if the tissue viability after exposure and post-treatment incubation is >50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 % aqueous solution

Duration of treatment / exposure:

60±1 minutes

Duration of post-treatment incubation (if applicable):

24 hours and 30 minutes

Number of replicates:

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

Irritation / corrosion parameter:

% tissue viability

Remarks:

test item

Run / experiment:

1

Value:

3.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

negative control

Run / experiment:

1

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

positive control

Run / experiment:

1

Value:

3.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Interpretation of results:

other: study cannot be used to decide on classification alone

Conclusions:

As a result of the available study, the test substance was identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1). Further information is required to distinguish between Category 1 and 2.

Executive summary:

A study was conducted to assess the skin irritation potential of the test item according to OECD Guideline No. 439. The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT. The test tissues were topically exposed to 30 µL of DPBS (negative control: NC), 30 µL of 5% aq. SDS solution (positive control: PC) or 30 µL of test item . All the treatments were maintained in triplicates. After 60 minutes of exposure the tissues were washed using DPBS. Later, the tissue inserts were blotted and transferred to fresh medium and incubated in an CO2 incubator for 24 hours and 30 minutes. After the incubation period (Day 1), the tissues were incubated for an additional 20 hours in the CO2 incubator. After this post-incubation period, the bottom of the tissue inserts was blotted and transferred into an MTT solution and incubated for 2 hours and 55 minutes. The optical density of the extracted formazan salt was afterwards measured in a 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated by entering OD values in the spread sheet provided by MatTek. The percentage of viability of the negative control, positive control and test item was 100±1.69, 3.7±0.03 and 3.8±0.22 respectively. As the percentage viability of the test item was not greater than 50% of the negative control, the test item is considered as “irritant”. The percentage of viability in the positive control (PC) was less than 50%, which shows the irritative potential of the positive control and the suitability of the test method. As the test method does not allow to distinguish between Category 1 and 2, further testing is required to exclude or confirm a corrosive property.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-11-08 to 2018-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

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 system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EpiSkinTM Small Model (EpiSkinTMSM), EPISKIN SNC Lyon, France, 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 corrosion testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
Supplier: SKINETHIC Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
Batch No.: 18-EKIN-045
Expiry date: 12 November 2018

Justification for test system used:

The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM)
- Tissue batch number: 18-EKIN-045

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (18-28°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
After the incubation time the EpiSkinTMSM units were rinsed thoroughly with approximately 25 mL PBS 1x solution to remove the test item from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
After the exposure of test item was terminated by rinsing with PBS, the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 15 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, ≥95% humidified atmosphere.
At the end of incubation with MTT a formazan extraction was undertaken:
A disk of epidermis was cut from the unit (this involves 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 of 500 μL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated overnight at room temperature protected from light for formazan extraction. At the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

NUMBER OF REPLICATE TISSUES: 2

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 < 35% (Sub category 1A) or if ean tissue viability is ≥ 35 % after 3 min exposure and < 35 % after 1 hour exposure OR
Mean tissue viability is ≥ 35 % after 1 hour exposure and < 35 % after 4 hours exposure (Sub-category 1B and 1C)
- The test item is considered as non-corrosive if the mean tissue viability is ≥ 35 % after 4 hours exposure

Acceptance criteria:
- The mean OD value of the two negative control tissues should be between 0.6 and 1.5.
- The acceptable percentage viability for positive control (each of two tissues) is 0 – 20 % (as per the manufactures specification, validated by ECVAM (Fentem et al, 1998)).
- In the range 20-100% viability and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

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.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of two replicates

Value:

66

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
- Direct-MTT reduction: no
- Colour interference with MTT: no

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

Table 1:OD values and cell viability percentages of the positive and negative control:

Controls	Optical Density (OD)		Viability (%)	Δ%
Negative Control: NaCl (9 g/L saline)	1	0.865	98	3.5
	2	0.896	102
	mean	0.881	100
Positive Control: Glacial acetic acid	1	0.004	1	0.3
	2	0.002	0
	mean	0.003	0

 

 

Table 2: OD values and viability percentages of the test item (including corrected values):

Test Item	Optical Density (OD)		Viability (%)	Δ%
Phosphoric acid, 2-ethylhexyl ester, sodium salt	1	0.581	66	0.8
	2	0.574	65
	mean	0.578	66

Remark:Δ%: The difference of viability between the two relating tissues

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions. In conclusion, the test item Phosphoric acid, 2-ethylhexyl ester, sodium salt can be classified as Non-corrosive.

Executive summary:

The EpiSkin- test of the test item Phosphoric acid, 2-ethylhexyl ester, sodium salt has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431, 29 July 2016.Disks of EPISKIN (two units / incubation time) were treated with test item and incubated for 4 hours (±10 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1°C in an incubator with 5±1 % CO₂ in a ≥ 95% humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after 4 hours of exposure. The average test item treated tissue viability was 66 % at 4 hours of exposure. The test item treated tissue viability was above 35 % of the mean negative control value after 4 hours of exposure. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid. The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions. In conclusion, the test item Phosphoric acid, 2-ethylhexyl ester, sodium salt can be classified as Non-corrosive.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

2017

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)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughter house (Chowdeshwari Chicken Center, Tumkur)
- Storage, temperature and transport conditions of ocular tissue: Eyes were enucleated as soon as possible after death and immersed in the Hank's Balanced Salt Solution (HBSS) with 10% antibiotics (Penicillin and Streptomycin) in a suitable container and were transported to the test facility by placing in cool packs.
- indication of any existing defects or lesions in ocular tissue samples: Upon arrival to the test facility, eyes were examined for defects including opacity, scratches and neovascularization. Only corneas free of such defects were used in the experiment.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Before the start of the experiment, opacity of empty cornea holders filled with MEM media were measured and the mean opacity value of the empty corneal holders obtained was considered as l0. Corneas free of defects were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in designated corneal holder's by placing the endothelial side of the cornea against the O-ring of the posterior chamber. The anterior chamber was placed over the cornea and both Chambers were joined together by tightening the chamber screws then the posterior and anterior chambers were filled with MEM without phenol red (Minimum Essential Medium supplemented with 1% Fetal Bovine Serum and 3% Penicillin and Streptomycin). The corneal holders were equilibrated at 32±1°C for one hour to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, MEM was replaced with fresh pre-warmed MEM without phenol red in both chambers of cornea holders after completion of equilibrium period. An opacity determination was performed on each of the corneas using an Opacitometer (BASF Opacitometer 2013-19). The opacity of each cornea was read against a MEM filled chamber, and the initial opacity reading thus determined was recorded us baseline opacity.
Opacity of each cornea was calculated by using a formula I0/I and opacity value was calculated for initial readouts (before treatment) by using the formula [I0/I-b)/a] where a=0.0251, b=0.9894 (Opacitometer specific empirically determined variables) I0 is the mean opacity value obtained for the empty corneal holders without corneas and with MEM, I is the individual opacity value of cornea. Corneas showing opacity greater than 7 opacity units after an initial 3-hour equilibration period were not used for the experiment,

TREATMENT METHOD: closed chamber

POST-EXPOSURE PERIOD: yes. 2 h

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the exposure period, the test item, negative and positive controls were removed from the anterior chamber and the epithelium was washed with EMEM containing phenol red until no visual evidence of the test item was observed. Finally the corneas were rinsed with MEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity was measured with the aid of an opacitometer. Opacity was then calculated using the formula mentioned in section 7,1.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea and positive control was calculated by subtracting the average change in opacity of die negative control corneas from the change in opacity of each test item treated and positive control cornea. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Run / experiment:

test item

Value:

70.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

negative control

Vehicle controls validity:

not examined

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: IVIS is not applicable for negative control

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control

Value:

121.3

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: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use the technical proficiency of the tost method was established by using proficiency chemicals under Bioneeds Study No.: BIO-TX 421, according to OECD Test Guideline No. 437. Proficiency chemicals are periodically tested in order to ensure the accuracy and reliability of the test method overtime (once in three years).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1 Summary of in vitro irritancy score (IVIS)

Group & Treatment	Mean change in opacity value	Mean corrected opacity value	Mean Corrected permeability value	IVIS value
Negative control	1.3	-	-	-
Positive control	97.5	93.19 ± 8.46	1.673 ± 0.00	121.3
Test item	51.0	49.70 ± 3.96	1.398 ± 0.175	70.7

 

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test according to OECD 437, the test item, induced an IVIS of 70.7 at 10 minutes of treatment. As the test item induced an IVIS >55, it is considered as severe irritant/causing serious eye damage to bovine cornea and classified as UN GHS category 1.

Executive summary:

The test item was evaluated for ocular corrosion or severe irritancy as per the OECD guideline for the testing of chemicals No. 437 "Bovine Corneal Opacity and Permeability Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals not requiring Classification for Eye Irritation or Serious Eye Damage", adopted October 2017.

Eyes of cattle were collected from a slaughter house by immersing them in the Hank's Balanced Salt Solution (HBSS) with antibiotics (penicillin and streptomycin) in a suitable container and transported to the test facility by placing on cool packs. Only eye balls free of defects were selected for the experiment. Empty cornea holder's opacity with pre-warmed Eagle's Minimum Essential Medium was measured and the mean opacity value obtained was determined as I0. Cornea holders with selected Corneas were equilibrated at 32±3 °C for 1 hour with Eagle's Minimum Essential Medium with 1% Fetal Bovine Scrum supplemented with 1% antibiotics and baseline opacity was recoiled for each cornea. Corneas with opacity units less than 7 were selected and used for the study and distributed for the treatment groups.

A volume of 750 µL of test item, negative (distilled water) and positive control (Ethanol) was introduced into anterior chamber in triplicates to die designated cornea holders and incubated at 32±1 °C for 10 minutes. Treated corneas were washed till no visual evidence of test item observed with EMEM containing phenol red and finally with EMEM without phenol red. The anterior chamber was then refilled with fresh EMEM without phenol red. Opacity was measured with the aid of opacitometer and permeability was determined spectrophotometrically at 490 nm (OD490) using 4 mg/mL sodium fluorescein, post incubation of 90 min at 32±1 °C.

The test item resulted in the mean corrected opacity and mean corrected permeability values of test item are 49.70 and 1.398, respectively. The in vitro Irritancy Score (IVIS) of test item resulted in 70.7, whereas the positive control resulted in mean corrected opacity and mean corrected permeability values of 93,19 and 1,673, respectively where the in vitro Irritancy Score (IVIS) of 121.3, indicating corrosion or severe irritancy to Bovine cornea.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation in vitro, OECD 439

A study was conducted to assess the skin irritation potential of the test item according to OECD Guideline No. 439. The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT. The test tissues were topically exposed to 30 µL of DPBS (negative control: NC), 30 µL of 5% aq. SDS solution (positive control: PC) or 30 µL of test item . All the treatments were maintained in triplicates. After 60 minutes of exposure the tissues were washed using DPBS. Later, the tissue inserts were blotted and transferred to fresh medium and incubated in an CO2 incubator for 24 hours and 30 minutes. After the incubation period (Day 1), the tissues were incubated for an additional 20 hours in the CO2 incubator. After this post-incubation period, the bottom of the tissue inserts was blotted and transferred into an MTT solution and incubated for 2 hours and 55 minutes. The optical density of the extracted formazan salt was afterwards measured in a 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated by entering OD values in the spread sheet provided by MatTek. The percentage of viability of the negative control, positive control and test item was 100±1.69, 3.7±0.03 and 3.8±0.22 respectively. As the percentage viability of the test item was not greater than 50% of the negative control, the test item is considered as “irritant”. The percentage of viability in the positive control (PC) was less than 50%, which shows the irritative potential of the positive control and the suitability of the test method. As the test method does not allow to distinguish between Category 1 and 2, further testing is required to exclude or confirm a corrosive property.

 

Skin corrosion in vitro, OECD 431

The EpiSkin- test of the test item Phosphoric acid, 2-ethylhexyl ester, sodium salt has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431, 29 July 2016.Disks of EPISKIN (two units / incubation time) were treated with test item and incubated for 4 hours (±10 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1°C in an incubator with 5±1 % CO₂in a ≥ 95% humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after 4 hours of exposure. The average test item treated tissue viability was 66 % at 4 hours of exposure. The test item treated tissue viability was above 35 % of the mean negative control value after 4 hours of exposure. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid. The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions.  

Skin irritation, supporting studies in vivo with aqueous solutions

In vivo studies on skin irritation potential with aqueous solutions of the test item are available. However, since the water moiety was up to ca. 50 % in the tested substances, the results of these studies are only taken into account as supporting information.

In conclusion, classification and labelling of the substance to be registered is done based on newer studies conducted with the substance. 

Skin irritation/corrosion observed in the acute dermal toxicity study

A study was conducted to assess the toxicity of test item when administered in a single dermal dose to rats at one or more defined dose levels.At first, the range-finding study was performed in one female Han:WISTrat. The starting dose was 200 mg/kg bw. The test item was applied in original form and left in contact with the skin for a 24 hours period. The observation period was three days (treatment day as Day 0 and post treatment period as Day 1 and Day 2). The in-life phase was terminated on Day 2 and the study was stopped, because unexpected corrosive signs were found on the treated skin surface. Moderate to severe erythema and other sign as whitish colouration were observed on Day 1, 1 hour after the patch removal. Severe erythema, open wound, crust and necrosis were detected on Day 2. No death and any systemic toxic sign were found. The body weight loss (approx. 4.2%) was observed between the treatment day and Day 2. External macroscopic changes as open wound, crust and necrosis were found on the treated area of the animal. The test item could not be classified into any toxicity category and was furthermore classified as corrosive to skin, into Category 1B, taking into account the 24 -hours exposure period of the rat, as this period is longer than the 4 hours exposure period applied in the vivo skin irritation study (OECD 404).

Eye irritation ex vivo, OECD 437

The test item was evaluated for ocular corrosion or severe irritancy following the OECD guideline for the testing of chemicals No. 437 "Bovine Corneal Opacity and Permeability Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals not requiring Classification for Eye Irritation or Serious Eye Damage", adopted October 2017.

Eyes of cattle were collected from a slaughter house by immersing them in the Hank's Balanced Salt Solution (HBSS) with antibiotics (penicillin and streptomycin) in a suitable container and transported to the test facility by placing on cool packs. Only eye balls free of defects were selected for the experiment. Empty cornea holder's opacity with pre-warmed Eagle's Minimum Essential Medium was measured and the mean opacity value obtained was determined as I0. Cornea holders with selected Corneas were equilibrated at 32±3 °C for 1 hour with Eagle's Minimum Essential Medium with 1% Fetal Bovine Scrum supplemented with 1% antibiotics and baseline opacity was recoiled for each cornea. Corneas with opacity units less than 7 were selected and used for the study and distributed for the treatment groups.

A volume of 750 µL of test item, negative (distilled water) and positive control (Ethanol) was introduced into anterior chamber in triplicates to die designated cornea holders and incubated at 32±1 °C for 10 minutes. Treated corneas were washed till no visual evidence of test item observed with EMEM containing phenol red and finally with EMEM without phenol red. The anterior chamber was then refilled with fresh EMEM without phenol red. Opacity was measured with the aid of opacitometer and permeability was determined spectrophotometrically at 490 nm (OD490) using 4 mg/mL sodium fluorescein, post incubation of 90 min at 32±1 °C.

The test item resulted in the mean corrected opacity and mean corrected permeability values of test item are 49.70 and 1.398, respectively. The in vitro Irritancy Score (IVIS) of test item resulted in 70.7, whereas the positive control resulted in mean corrected opacity and mean corrected permeability values of 93,19 and 1,673, respectively where the in vitro Irritancy Score (IVIS) of 121.3, indicating corrosion or severe irritancy to Bovine cornea.

 

Eye irritation, supporting studies in vivo

In vivo studies on eye irritation potential with aqueous solutions of the test item are available. However, since the water moiety was up to ca. 50 % in the tested substances, the results of theses studies are only taken into account as supporting information.

In conclusion, classification and labelling of the substance to be registered is done based on the in vitro test conducted with this substance. This approach is further considered to reflect the worst case as the substance is classified as severely eye damaging Cat 1.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item is classified and labelled as skin corrosive Cat. 1B and eye damaging Cat 1 and is labelled with H314: "Causes severe skin burns and eye damage") according to Regulation (EC) No 1272/2008 (CLP).