[No public or meaningful name is available]

Endpoint summary

Description of key information

Following a weight of evidence approach including results of two in vitro studies on skin irritation/corrosion according to OECD 439 and 431, respectively, the test item is considered to be corrosive to skin.

Based on a weight of evidence approach with results obtained in OECD 437 and OECD 492 compliant studies, the test item 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 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

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:

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.

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

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

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

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

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

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

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.

    

 

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).

Reference 2

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 The test item 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:

6.3

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:

6.9

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±2.25, 6.9±0.16 and 6.3±0.02 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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:

2018-04-03 to 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:

82.3

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 not applicable for negative control

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control

Value:

116.8

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	93.6	92.30 ± 6.37	1.635 ± 0.009	116.8
Test item	60.6	59.30 ± 3.75	1.536 ± 0.103	82.3

 

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 82.3 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 59.30 and 1.536, respectively. The in vitro Irritancy Score (IVIS) of test item resulted in 82.3, whereas the positive control resulted in mean corrected opacity and mean corrected permeability values of 92,30 and 1,635, respectively where the in vitro Irritancy Score (IVIS) of 116.8, indicating corrosion or severe irritancy to Bovine cornea.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-04-05 to 2018-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human EpiOcular™ Model (OCL-200-EIT)
- Tissue batch number: 27030

FUNCTIONAL MODEL CONDITIONS
- Viability: The tissue viability was within the acceptable range.
- Barrier function: The barrier function was within the acceptable range.
- Morphology: The morphology was within the acceptable range.
- Contamination: No contaminations were determined.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

VEHICLE
- Amount applied: 50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

12 min (post-exposure soak) + 120 min (post-exposure incubation)

Number of animals or in vitro replicates:

2 replicates per test item, negative control and positive control, respectively

Details on study design:

- RhCE tissue construct used, including batch number: Reconstructed Human EpiOcular™ Tissue, Batch number: 27030
- Doses of test chemical and control substances used: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: exposure: 30 min, 37 °C; post-exposure immersion: 12 min, room temperature; post-exposure incubation: 120 min, 37 °C
- Number of tissue replicates used per test chemical and controls: positive control: 2 tissue replicates; negative control: 2 tissue replicates
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan: determination of the optical density (OD) in a 96-well plate using a plate reader at a wavelength of 570 nm
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
The test item is labeled as “non-irritant” (UN GHS No Category), if the test item treated percent tissue viability determined by MTT assay is >60% relative to the negative control treated tissue viability.
No prediction can be made if the percent tissue viability determined by MTT assay is ≤60% relative to the negative control treated tissue viability.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: 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: mean viability %

Run / experiment:

1-2

Value:

2.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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

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

 

Refer Appendix-1

Treatment		OD	Viability (%)	Viability difference between tissues	Classification
Negative Control (Sterile water)	Mean	1.056	100.00	0.07	NI
	±SD	0.001	0.14
	n	2	2	2
Positive Control (Methyl acetate)	Mean	0.022	2.1	0.02	I
	±SD	0.001	0.05
	n	2	2	2
Test Item [Phosphoric acid,   2-etylhexyl ester, NH4+ salt (Primasol NF)]	Mean	0.031	2.9	0.00	I
	±SD	0.001	0.05
	n	2	2	2

NI = Non Irritant; I = Irritant; n = No. of tissues

Interpretation of results:

other: no prediction can be made based on the study outcome. Result has to be evaluated in a weight of evidence approach including further tests.

Conclusions:

Based on the results obtained under the conditions of this study, the test item has to be categorized as irritant or corrosive to the eye (UN GHS Category 1 or Category 2) as the mean percentage tissue viability is less than 60% of the negative control.

Executive summary:

The objective of this study was to evaluate the in vitro eye irritation potential of the test item using EpiOcular™ model (OCL-200-EIT) as per the OECD guideline for the testing of chemicals, Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage 492, adopted on 9th October 2017.

The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. OD reading was <0.08.

Tissues were equilibrated for 15 minutes at room temperature and then transferred to 6-well plates prefilled with 1.0 mL of assay medium and incubated in CO2 incubator at 37±1°C and 5±1% CO2 for 16 hours.

Tissues were pre-wetted with 20 µL DPBS and tapped to ensure that DPBS spreads all over the tissues surface. All the treatments were maintained in duplicates. The tissues were incubated at standard culture conditions for 30 minutes. Post 30 minutes of incubation, 50 µL each of test item, negative control and positive control were dispensed directly atop EpiOcular™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 32 minutes. At the end of treatment time with test item, negative control and positive control, tissue inserts were dipped into the first tubes of DPBS, swirled in a circular motion in the liquid for 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 tube. The cultures were then rinsed in the second and third tubes of DPBS. After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed assay medium in a prelabeled 12-well plate for 12 minutes immersion incubation (Post-Soak) at room temperature which was intended to remove any test item absorbed. 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 insert was blotted onto the absorbent material, and transferred to the appropriate well of the prelabeled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 120 minutes in CO2 incubator at 37±1°C and 5±1% CO2 (Post-treatment Incubation). Post 120 minutes of incubation with the assay medium, 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 170 minutes 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 prelabeled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface. The plates are sealed with parafilm and stored overnight at 2 to 8°C in the dark. The plates were removed and placed on an orbital plate shaker and shaken for 2 hours at room temperature for extraction process. At the end of the extraction period, the tissue was pierced with an injection needle and the liquid within each insert was decanted into the well from which it was taken. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated. Mean percentage viability of negative control, positive control and test item were 100±0.14, 2.1±0.05 and 2.9±0.05 respectively. As the mean percentage viability of test item was less than 60% of the negative control considered as irritant, similarly the percentage viability of positive control is less than 60% of negative control clearly represents the irritation potential of PC.

 

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±2.25, 6.9±0.16 and 6.3±0.02 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 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).

 

 

Eye irritation ex vivo, OECD 437

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 59.30 and 1.536, respectively. The in vitro Irritancy Score (IVIS) of test item resulted in 82.3, whereas the positive control resulted in mean corrected opacity and mean corrected permeability values of 92,30 and 1,635, respectively where the in vitro Irritancy Score (IVIS) of 116.8, indicating corrosion or severe irritancy to Bovine cornea.

Eye irritation in vitro, OECD 492

The objective of this study was to evaluate the in vitro eye irritation potential of the test item using EpiOcular™ model (OCL-200-EIT) as per the OECD guideline for the testing of chemicals, Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage 492, adopted on 9th October 2017.

The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. OD reading was <0.08.

Tissues were equilibrated for 15 minutes at room temperature and then transferred to 6-well plates prefilled with 1.0 mL of assay medium and incubated in CO2 incubator at 37±1°C and 5±1% CO2 for 16 hours.

Tissues were pre-wetted with 20 µL DPBS and tapped to ensure that DPBS spreads all over the tissues surface. All the treatments were maintained in duplicates. The tissues were incubated at standard culture conditions for 30 minutes. Post 30 minutes of incubation, 50 µL each of test item, negative control and positive control were dispensed directly atop EpiOcular™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 32 minutes. At the end of treatment time with test item, negative control and positive control, tissue inserts were dipped into the first tubes of DPBS, swirled in a circular motion in the liquid for 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 tube. The cultures were then rinsed in the second and third tubes of DPBS. After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed assay medium in a prelabeled 12-well plate for 12 minutes immersion incubation (Post-Soak) at room temperature which was intended to remove any test item absorbed. 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 insert was blotted onto the absorbent material, and transferred to the appropriate well of the prelabeled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 120 minutes in CO2 incubator at 37±1°C and 5±1% CO2 (Post-treatment Incubation). Post 120 minutes of incubation with the assay medium, 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 170 minutes 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 prelabeled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface. The plates are sealed with parafilm and stored overnight at 2 to 8 °C in the dark. The plates were removed and placed on an orbital plate shaker and shaken for 2 hours at room temperature for extraction process. At the end of the extraction period, the tissue was pierced with an injection needle and the liquid within each insert was decanted into the well from which it was taken. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated. Mean percentage viability of negative control, positive control and test item were 100±0.14, 2.1±0.05 and 2.9±0.05 respectively. As the mean percentage viability of test item was less than 60% of the negative control considered as irritant, similarly the percentage viability of positive control is less than 60% of negative control clearly represents the irritation potential of PC.

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. 1 (sub-categories 1B and 1C, H314: "Causes severe skin burns and eye damage") and eye damaging Cat 1 (H318: "Causes serious eye damage") according to Regulation (EC) No 1272/2008 (CLP).