Phosphoric acid, butyl ester, sodium salt

Endpoint summary

Description of key information

Based on a weight of evidence approach the test substance is considered to be skin irritanting (UN GHS Category 2).

Based on a weight of evidence approach the test substance is considered to be eye damaging (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 vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1978

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

not specified

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

not specified

Controls:

not required

Observation period:

Readings were made 24 and 72 hours after application

Number of animals:

6

Irritation parameter:

erythema score

Remarks:

intact skin

Basis:

animal: #1, #3

Time point:

24 h

Score:

2

Max. score:

8

Reversibility:

not fully reversible within: 72 h

Remarks:

erythema score: 1

Irritation parameter:

erythema score

Remarks:

intact skin

Basis:

animal: #2, #4 - #6

Time point:

24 h

Score:

2

Max. score:

8

Reversibility:

not fully reversible within: 72 h

Remarks:

erythema score: 1

Irritation parameter:

erythema score

Remarks:

sore skin

Basis:

animal #1

Time point:

24 h

Score:

3

Max. score:

8

Reversibility:

not fully reversible within: 72 h

Remarks:

erythema score: 1

Irritation parameter:

erythema score

Remarks:

sore skin

Basis:

animal: #2 - #6

Time point:

24 h

Score:

2

Max. score:

8

Reversibility:

not fully reversible within: 72 h

Remarks:

erythema score: 1

Irritation parameter:

erythema score

Remarks:

intact and sore skin

Basis:

animal: #1 - #6

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

erythema score

Remarks:

intakt skin

Basis:

animal: #1 - #6

Time point:

72 h

Score:

1

Max. score:

8

Irritation parameter:

erythema score

Remarks:

sore skin

Basis:

animal: #1 - #6

Time point:

72 h

Score:

1

Max. score:

8

Irritation parameter:

edema score

Remarks:

intact skin

Basis:

animal: #1, #3

Time point:

24 h

Score:

0

Max. score:

8

Irritation parameter:

edema score

Remarks:

intact skin

Basis:

animal: #2, #4 - #6

Time point:

24 h

Score:

1

Max. score:

8

Reversibility:

fully reversible within: 72 h

Irritation parameter:

edema score

Remarks:

sore skin

Basis:

animal: #1, #3

Time point:

24 h

Score:

1

Max. score:

8

Reversibility:

fully reversible within: 72 h

Irritation parameter:

edema score

Remarks:

sore skin

Basis:

animal: #2, #5

Time point:

24 h

Score:

1

Max. score:

8

Reversibility:

not fully reversible within: 72 h

Remarks:

edema score: 1

Irritation parameter:

edema score

Remarks:

sore skin

Basis:

animal #4

Time point:

24 h

Score:

2

Max. score:

8

Reversibility:

not fully reversible within: 72 h

Remarks:

edema score: 2

Irritation parameter:

edema score

Remarks:

sore skin

Basis:

animal #6

Time point:

24 h

Score:

1

Max. score:

8

Reversibility:

not fully reversible within: 72 h

Remarks:

edema score: 72 h

Irritation parameter:

edema score

Remarks:

intact and sore skin

Basis:

animal: #1 - #6

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Remarks:

intact skin

Basis:

animal: #1 - #6

Time point:

72 h

Score:

0

Max. score:

8

Irritation parameter:

edema score

Remarks:

sore skin

Basis:

animal: #1, #3

Time point:

72 h

Score:

0

Max. score:

8

Irritation parameter:

edema score

Remarks:

sore skin

Basis:

animal: #2, #5

Time point:

72 h

Score:

1

Max. score:

8

Irritation parameter:

edema score

Remarks:

sore skin

Basis:

animal: #4, #6

Time point:

72 h

Score:

2

Max. score:

8

Irritant / corrosive response data:

Erythema were mainly seen after 24 hours. Based on the applied scoring system, the substance was found to be mildly irritating to skin.

Interpretation of results:

other: Due to the limited documentation the study cannot be used for classification.

Conclusions:

Signs of mild irritation were seen in the study.

Executive summary:

A study was performed to assess the irritation of the test material to the skin of six rabbits. As a result, erythema and edema were seen in all animals. Erythema were not reversible within 72 hours. Based on the applied scoring system, the substance was found to be mildly irritating to skin, but due to the limited documentation no conclusion can be drawn on classification.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, U.K
- Age at study initiation: 12-16 weeks
- Weight at study initiation: 2.47-2.7 kg
- Housing: The animals were individually housed in suspended metal cages.
- Diet: ad libitum, RABMA Rabbit Diet, Special Diet Services Ltd., Witham, Essex, UK)
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 58-64
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

other: shorn

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of irritation.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm on the back
- Type of wrap if used: gauze patch secured with surgical adhesive tape, the trunk of each rabbit was wrapped in an elastic corset

REMOVAL OF TEST SUBSTANCE
- Washing: gentle swabbing with cotton wool soaked in distilled water
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
1, 24, 48, 72 hrs after patch removal

SCORING SYSTEM:
- according to Draize

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Very slight erythema was noted at all treated skin sites one hour after patch removal and at one treated skin site at the 24-hour observation. The reaction extended up to 1.5 cm beyond one treatment site one hour after patch removal. Very slight oedema was noted at two treated skin sites one hour after patch removal. Alt treated skin sites appeared normal at the 48-hour observation.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results the test item is not corrosive to skin and it is not to be calssfied for skin irritation.

Executive summary:

The study was performed to assess the irritancy potential of the test material following a single, 4-hour, semi-occluded application to the intact rabbit skin. The method used followed OECD Guideline No. 404. Three female New Zealand White rabbits were used. A single 4-hour, semi-occluded application of the test material to the intact skin produced very slight erythema and very slight oedema (score 1 of 4 each). All treated skin sites appeared normal at the 48-hour observation. Based on these results, it was concluded that the test item is not to be classified for skin irritation.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2002

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Type of coverage:

semiocclusive

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

3-minute and 1-hour (pretest), 4 hours (main test)

Observation period:

Approximately one hour following the removal of the patches, and 24,48 and 72 hours later, the test sites were examined for evidence of irritation.

Number of animals:

1 (pretest), 3 further animals (main test)

Details on study design:

TEST SITE
intact skin

OBSERVATION TIME POINTS
1, 24, 48, 72 hrs after patch removal

SCORING SYSTEM:
- according to Draize

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Very slight to well-defined erythema was noted at all treated skin sites one hour after patch removal with very slight erythema noted at two treated skin sites at the 24-hour observation. Slight oedema was noted at two treated skin sites one hour after patch removal. All findings were reversible within 48 hours.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance does not meet criteria to be classified as skin irritant.

Executive summary:

A study was performed to assess the irritation of the test material to the skin of the New Zealand White rabbit. 3-minute and 1-hour semi-occluded applications (0.5 mL) of the test material were administered to the intact skin of one rabbit. Skin reactions were recorded 1, 24, 48 and72 hours after administration. A single 4-hour, semi-occluded application (0.5 mL) of the test material was administered to the intact skin of three rabbits. Skin reactions were recorded 1, 24, 48 and 72 hours after administration. Very slight to well-defined erythema was noted at all treated skin sites one hour after patch removal with very slight erythema noted at two treated skin sites at the 24-hour observation. Slight oedema was noted at two treated skin sites one hour after patch removal. The mean scores (over 24, 48, 72 h readings) per animal were all 0 for edema, and 0.3 (two animals) and 0(one animal) for erythema formation. All findings were reversible within 48 hours. Based on the results the test substance does not meet criteria to be classified as skin irritant.

Reference 4

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
- Temperature of post-treatment incubation (if applicable):

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: 25 mg

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:

4.7

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:

6.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Interpretation of results:

other: Category 1 (irritant) or Category 2 (corrosive) based on GHS criteria

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 dstinguish 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 25 mg 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±0.90, 6.1±0.34 and 4.7±0.05 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 5

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-05-17

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

Cell source:

other: 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 corrosion.

Vehicle:

unchanged (no vehicle)

Details on test system:

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

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsing with sterile PBS, fill and empty insert 20 times in a constant soft stream of (1xPBS)
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h +/- 5 min
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: approved
- Barrier function: approved
- Morphology: well
- Contamination: none

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% and 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 min and 1 h

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 minutes exposure

Run / experiment:

tissue 1

Value:

92.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 minutes exposure

Run / experiment:

tissue 2

Value:

91

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

1 hour exposure

Run / experiment:

tissue 1

Value:

81.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

1 hour exposure

Run / experiment:

tissue 2

Value:

90.1

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

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use 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

Interpretation of results:

other: not Category 1 (corrosive) based on GHS criteria

Conclusions:

The test item is categorised as non-corrosive to Reconstructed Human Epidermis (RhE) as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.

Executive summary:

The objective of study was to evaluate the for 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 developed any colour when dissolved in distilled water/isopropanol and considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. After receipt of the tissues, visual inspection was done to verify the defects, as there were no tissue defects, air bubble and 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 items were exposed for 1 hour and 3 minutes separately. All the treatments were maintained in duplicates. For the 3 minute and for the 1 hour treatment, 25 mg of test item + 25 µL distilled water, 50 µL of negative control and 50 µL of positive control were dispensed directly atop Epiderm™ tissues at 1 minute intervals, respectively. The tissues were incubated at standard culture conditions for 3 minute or 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 prelabeled 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 5 hours and 10 minutes (for 1 hour exposure) and 4 hours and 48 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 becomes 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±1.0 %, 6.1±0.1 % and 92±1.8 %, respectively. As the percentage viability of test item was greater than 50% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control clearly represents the irritation potential of positive control.

For 1 hour exposure, percentage viability of negative control, positive control and test item was 100±2.4 %, 6.0±0.6 % and 85.9±8.4 %, respectively. As the percentage viability of test item was greater than 15% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control clearly represents the irritation potential of positive control.

Based on the results obtained under the laboratory testing conditions, the test item is categorised as non-corrosive to Reconstructed Human Epidermis (RhE) as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1978

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

not specified

GLP compliance:

not specified

Species:

rabbit

Strain:

not specified

Vehicle:

not specified

Controls:

not required

Duration of treatment / exposure:

single exposure

Observation period (in vivo):

the eyes were examined after 1, 2 and 3 days

Number of animals or in vitro replicates:

6 animals

Irritation parameter:

conjunctivae score

Remarks:

mean

Basis:

animal #1

Time point:

24/48/72 h

Score:

10

Max. score:

56

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

mean

Basis:

animal #2

Time point:

24/48/72 h

Score:

6.7

Max. score:

56

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

mean

Basis:

animal #3

Time point:

24/48/72 h

Score:

12

Max. score:

56

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

mean

Basis:

animal #4

Time point:

24/48/72 h

Score:

8.7

Max. score:

56

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

mean

Basis:

animal #5

Time point:

24/48/72 h

Score:

6

Max. score:

56

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

mean

Basis:

animal #6

Time point:

24/48/72 h

Score:

13.3

Max. score:

56

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Remarks:

mean

Basis:

animal #1

Time point:

24/48/72 h

Score:

20

Max. score:

56

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Remarks:

mean

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

56

Irritation parameter:

cornea opacity score

Remarks:

mean

Basis:

animal #3

Time point:

24/48/72 h

Score:

20

Max. score:

56

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Remarks:

mean

Basis:

animal #4

Time point:

24/48/72 h

Score:

6.7

Max. score:

56

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Remarks:

mean

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Remarks:

mean

Basis:

animal #6

Time point:

24/48/72 h

Score:

20

Max. score:

56

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

iris score

Remarks:

mean

Basis:

animal: #1 - #6

Time point:

24/48/72 h

Score:

0

Max. score:

56

Irritation parameter:

chemosis score

Basis:

animal: #1 - #6

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritant / corrosive response data:

Conjunctiva and cornea findings were not reversible within 72 hours.

Interpretation of results:

other: The study cannot be used for assessment due to its insufficient documentation.

Conclusions:

The tested substance caused weak signs of irritation to the eyes, but based on the available data no conclusion on classification can be made.

Executive summary:

A study was performed to assess the irritation of the test material to the eyes. Six rabbits were used. As a result, conjunctiva and cornea findings were made which were not reversible within 72 hours. Based on the applied scoring system, the substance was found to cause weak signs of irritation to the eyes. Due to the limited documentation no conclusion can be made on classification.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, UK
- Age at study initiation: 12-16 weeks
- Weight at study initiation: 2.7 - 3.24 kg
- Housing: The animals were individually housed in suspended metal cages.
- Diet: ad libitum, ABMA Rabbit Diet, Special Diet Services Ltd., Witham, Essex, UK
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22
- Humidity (%): 59-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL

Duration of treatment / exposure:

single treatment, the upper and lower eyelids were held together for about one second immediately after instillation, to prevent loss of the test material, and then released

Observation period (in vivo):

Assessment of ocular damage/ircitation was made approximately 1 hour and 24, 48 and 72 hours following treatment.

Number of animals or in vitro replicates:

1 animals wa sinitially treated, afterwards two more animals were treated

Details on study design:

In order to minimise pain on instillation of the test material, one drop of local anaesthetic ("Ophthaine", 0.5% proxymetacaine hydrochloride, E.R. Squibb & Sons, Limited Hounslow, Middlesex, UK) was instilled into both eyes of the animals 1 - 2 minutes before treatment.

SCORING SYSTEM: according to Draize

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Diffuse corneal opacity was noted in one treated eye at the 24-hour observation. No other corneal effects were noted. No iridial effects were noted during the study. Minimal conjunctival irritation was noted in all treated eyes one hour after treatment and in two treated eyes at the 24-hour observation. All treated eyes appeared normal 24 - 48 hours after treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

The test material was considered as non-irritant to the rabbits' eyes.

Executive summary:

The study was performed according to OECD Guideline No. 405 to assess the irritancy potential of the test material following a single application to the eye. New Zealand White rabbits were used in the study. One rabbit was initially treated. A volume of 0.1 mL of the test material was instilled into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after instillation, to prevent loss of the test material, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test material, an assessment of the initial pain reaction was made. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated. In order to minimise pain on instillation of the test material, one drop of local anaesthetic ("Ophthaine", 0.5% proxymetacaine hydrochloride, E.R. Squibb & Sons, Limited Hounslow, Middlesex, UK) was instilled into both eyes of these animals 1 - 2 minutes before treatment. Diffuse corneal opacity was noted in one treated eye at the 24-hour observation. No other corneal effects were noted. No iridial effects were noted during the study. Minimal conjunctival irritation was noted in all treated eyes one hour after treatment and in two treated eyes at the 24-hour observation. Treated eyes appeared normal 24 - 48 hours after treatment. Based on these results, the test material was considered as non-irritant to the rabbits’ eyes.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2007-03-27 to 2007-04-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2002

Deviations:

not specified

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL

Duration of treatment / exposure:

one single application

Observation period (in vivo):

Ocular reactions were recorded 1, 24, 48 and 72 hours and 7 days after administration.

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:
Cornea opacity score: 0-4
Iris score: 0-2
Conjunctivae score: 0-3
Chemosis score: 0-4

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 72 h

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

The test material produced iridial inflammation and moderate conjunctival irritation. One treated eye appeared normal at the 72-hour observation and the remaining two treated eyes appeared normal at the 7-day observation.

Interpretation of results:

GHS criteria not met

Conclusions:

The test material is not to be classified for eye irriation.

Executive summary:

The study was performed according to OECD Guideline No. 405 to assess the irritancy potential of the test material following a single application to the eye in New Zealand White rabbits. A single application of 0.1 mL of the test material was administered to the non irrigated eye of three rabbits. Ocular reactions were recorded 1, 24, 48 and 72 hours and 7 days after administration. The test material produced iridial inflammation and moderate conjunctival irritation. One treated eye appeared normal at the 72-hour observation and the remaining two treated eyes appeared normal at the 7-day observation. The test material produced iridial inflammation and moderate conjunctival irritation. One treated eye appeared normal at the 72-hour observation and the remaining two treated eyes appeared normal at the 7-day observation. Based on the results, the test material is not to be classified for eye irritation.

Reference 4

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-04-04

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:

9th October 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:

9th December 2010

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: slaughter house (Choudeshwari chicken centre, Tumkur)
- Characteristics of donor animals: less than 5 years old
- Storage, temperature and transport conditions of ocular tissue: To prevent exposure of the eyes to potentially irritant substances, the heads of the animals were not rinsed with detergent. 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.

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:

The corneas were exposed to the test item for 10 minutes. A similar procedure was followed for negative and positive controls.

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS
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.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
distilled water

POSITIVE CONTROL USED
ethanol

APPLICATION DOSE AND EXPOSURE TIME
750 µL, 10 min exposure

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes, 2 h

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
A test item was classified based on the obtained IVIS and categorized as mentioned below:
- a test item with an IVIS score ≤ 3 is considered as not requiring classification for eye irritation or serious eye damage according to UN GHS.
- a test item with an IVIS score >3 and ≤ 55 is considered as equivocal, subsequently testing with any other adequate method remains at the discretion of the sponsor.
- a test item with an IVIS score > 55 is considered as severe irritant causing serious eye damage and classified as UN GHS category 1 without further testing.

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

1-3

Value:

83.7

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: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use the technical proficiency of the test method was established by using proficiency chemicals, according to OECD Test Guideline No. 437.

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

Interpretation of results:

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

Conclusions:

The test item induced an IVIS >55 and is considered as severe irritant/causing serious eye damage to bovine cornea (UN GHS Category 1).

Executive summary:

The test item was evaluated for ocular corrosivity or severe irritancy as per OECD guideline 437. 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. 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±1 ºC for 1 hour with Eagle’s Minimum Essential Medium with 1% Fetal Bovine Serum supplemented with 1% antibiotics and baseline opacity was recorded 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 20% w/v test item, vehicle (distilled water) and positive control (20% w/v imidazole) was introduced into anterior chamber in triplicates to the designated cornea holders and incubated at 32±1 ºC for 4 hours. 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 an opacitometer and permeability was determined spectrophotometrically at 490 nm (OD490) using 5 mg/mL sodium fluorescein, post incubation of 90 min at 32±1 ºC.

The test item resulted in a mean corrected opacity and mean corrected permeability values of 59.8 and 1.59, respectively. The in vitro Irritancy Score (IVIS) of test item was 83.7. The positive control resulted in mean corrected opacity and mean corrected permeability values of 97.69 and 1.624, respectively. The in vitro Irritancy Score (IVIS) of the positive control was 122.1, indicating corrosivity or severe irritancy to bovine cornea.

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test, the test item induced an IVIS of 83.7 after 4 hours of treatment. The results indicated an appreciable increase in the endpoint “opacity” and “permeability”. 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

In vivo studies:

Several studies are available which were assessed based on a weight of evidence-approach.

One study was performed to assess the irritancy potential of the test material (aqueous solution, 80 % water) following a single, 4-hour, semi-occluded application to the intact rabbit skin. The method used followed OECD guideline 404. Three female New Zealand White rabbits were exposed. A single 4-hour, semi-occluded application of the test material to the intact skin produced very slight erythema and very slight oedema (score 1 of 4 each). All treated skin sites appeared normal at the 48-hour observation. Based on these results, it was concluded that the test item is not to be classified for skin irritation. 

In another study the test material (aqueous solution, 45 % water) was also tested on the skin of the New Zealand White rabbit. 3-minute and 1-hour semi-occluded applications (0.5 mL) of the test material were administered to the intact skin of one rabbit. Skin reactions were recorded 1, 24, 48 and72 hours after administration. A single 4-hour, semi-occluded application (0.5 mL) of the test material was administered to the intact skin of three rabbits. Skin reactions were recorded 1, 24, 48 and 72 hours after administration. Very slight to well-defined erythema was noted at all treated skin sites one hour after patch removal with very slight erythema noted at two treated skin sites at the 24-hour observation. Slight oedema was noted at two treated skin sites one hour after patch removal. The mean scores (over 24, 48, 72 h readings) per animal were all 0 for edema, and 0.3 (two animals) and 0(one animal) for erythema formation. All findings were reversible within 48 hours. Based on the results the test substance does not meet criteria to be classified as skin irritant. 

A further study was performed to assess the irritation of the test material (aqueous solution, 81.5 % water) to the skin of six rabbits. Only limited documentation of this study is available. As a result of the study, erythema and edema were seen in all animals. Erythema were not reversible within 72 hours. Due the limited documentation no conclusion can be drawn on classification.

 

In vitro studies:

A study was conducted to assess the skin irritation potential of the test item according to OECD guideline 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 25 mg 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±0.90 %, 6.1±0.34 % and 4.7±0.05 %, 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” (UN GHS Cat. 2) or "corrosive" (UN GHS Cat. 1). The percentage of viability in the positive control (PC) was less than 50%, which showed 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.

Therefore, an in vitro skin corrosion study according to OECD guideline 431 has been performed. The objective of study was to evaluate the for 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 developed any colour when dissolved in distilled water/isopropanol and considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. After receipt of the tissues, visual inspection was done to verify the defects, as there were no tissue defects, air bubble and 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 items were exposed for 1 hour and 3 minutes separately. All the treatments were maintained in duplicates. For the 3 minute and for the 1 hour treatment, 25 mg of test item + 25 µL distilled water, 50 µL of negative control and 50 µL of positive control were dispensed directly atop Epiderm™ tissues at 1 minute intervals, respectively. The tissues were incubated at standard culture conditions for 3 minute or 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 prelabeled 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 5 hours and 10 minutes (for 1 hour exposure) and 4 hours and 48 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 becomes 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±1.0 %, 6.1±0.1 % and 92±1.8 %, respectively. As the percentage viability of test item was greater than 50% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control clearly represents the irritation potential of positive control.

For 1 hour exposure, percentage viability of negative control, positive control and test item was 100±2.4 %, 6.0±0.6 % and 85.9±8.4 %, respectively. As the percentage viability of test item was greater than 15% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control clearly represents the irritation potential of positive control. Based on the results obtained under the laboratory testing conditions, the test item is categorised as non-corrosive to Reconstructed Human Epidermis (RhE) as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.

Conclusion:

All in vivo studies were performed with test items with minor concentration/purity. Therefore, the classification was made based on in vitro studies performed with the pure test item (100%). As worst case the test substance is considered to be skin irritating (UN GHS Category 2).

Eyes:

In vivo studies:

Several studies are available which were assessed based on a weight of evidence-approach.

One study was performed according to OECD guideline 405 to assess the irritancy potential of the test material (aqueous solution, 45 % water) following a single application to the eye in New Zealand White rabbits. A single application of 0.1 mL of the test material was administered to the non irrigated eye of three rabbits. Ocular reactions were recorded 1, 24, 48 and 72 hours and 7 days after administration. The test material produced iridial inflammation and moderate conjunctival irritation. One treated eye appeared normal at the 72-hour observation and the remaining two treated eyes appeared normal at the 7-day observation. The test material produced iridial inflammation and moderate conjunctival irritation. One treated eye appeared normal at the 72-hour observation and the remaining two treated eyes appeared normal at the 7-day observation. Based on the results, the test material is not to be classified for eye irritation.

Another study was performed according to OECD guideline 405 to assess the irritancy potential of the test material (aqueous solution, 80 % water) following a single application to the eye. New Zealand White rabbits were used in the study. One rabbit was initially treated. A volume of 0.1 mL of the test material was instilled into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after instillation, to prevent loss of the test material, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test material, an assessment of the initial pain reaction was made. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated. In order to minimise pain on instillation of the test material, one drop of local anaesthetic ("Ophthaine", 0.5% proxymetacaine hydrochloride, E.R. Squibb & Sons, Limited Hounslow, Middlesex, UK) was instilled into both eyes of these animals 1 - 2 minutes before treatment. Diffuse corneal opacity was noted in one treated eye at the 24-hour observation. No other corneal effects were noted. No iridial effects were noted during the study. Minimal conjunctival irritation was noted in all treated eyes one hour after treatment and in two treated eyes at the 24-hour observation. Treated eyes appeared normal 24 - 48 hours after treatment. Based on these results, the test material was considered as non-irritant to the rabbits’ eyes.

A further study was conducted with the test item as aqueous solution containing 81.5 % water to assess the irritation to the eyes. Only limited documentation of this study is available. Six rabbits were used. As a result, conjunctiva and cornea findings were made which were not reversible within 72 hours. Based on the applied scoring system, the substance was found to cause weak signs of irritation to the eyes. Due to the limited documentation no conclusion can be made on classification.

In vitro studies:

The test item was evaluated for ocular corrosivity or severe irritancy as per OECD guideline 437. 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. 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±1 ºC for 1 hour with Eagle’s Minimum Essential Medium with 1% Fetal Bovine Serum supplemented with 1% antibiotics and baseline opacity was recorded 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 20% w/v test item, vehicle (distilled water) and positive control (20% w/v imidazole) was introduced into anterior chamber in triplicates to the designated cornea holders and incubated at 32±1 ºC for 4 hours. 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 an opacitometer and permeability was determined spectrophotometrically at 490 nm (OD490) using 5 mg/mL sodium fluorescein, post incubation of 90 min at 32±1 ºC.

The test item resulted in a mean corrected opacity and mean corrected permeability values of 59.8 and 1.59, respectively. The in vitro Irritancy Score (IVIS) of test item was 83.7. The positive control resulted in mean corrected opacity and mean corrected permeability values of 97.69 and 1.624, respectively. The in vitro Irritancy Score (IVIS) of the positive control was 122.1, indicating corrosivity or severe irritancy to bovine cornea.

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test, the test item induced an IVIS of 83.7 after 4 hours of treatment. The results indicated an appreciable increase in the endpoint “opacity” and “permeability”. 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.

Conclusion:

All in vivo studies were performed with test items with minor concentration/purity. Therefore, the classification was made based on an in vitro study performed with the pure test item (100%). Based on a weight of evidence approach the test substance is considered to be eye damaging (UN GHS Category 1).

 

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on this data, the substance is considered to be classified for skin irritation (Cat. 2, H315) and eye irritation (Cat. 1, H318) under Regulation (EC) No 1272/2008, as amended for the twelth time in Regulation (EU) No 2019/521.