4-bromobenzyl alcohol

Endpoint summary

Description of key information

Based on the results of an in vitro skin corrosion study according to OECD 431, the test substance did not show skin corrosion properties (reference 7.3.1 -1).

Based on the results of an in vitro skin irritation study according to OECD 439, the test substance did not show skin irritation properties (reference 7.3.1 -2).

Based on the results of an in vitro eye irritation study according to OECD 437, the test item is not considered to have eye damaging potential (UN GHS: Category 1). No prediction can be made for eye damaging potential UN GHS Category 2 or no classification for eye damaging potential (reference 7.3.2-1).

Based on the results of an in vitro eye irritation study according to OECD 492, the test item is not considered to not require classification (UN GHS: No Category). No prediction on classification UN GHS Category 1 or 2 can be made (reference 7.3.2 -2)

Based on the results of the two eye irritation studies, the substance is considered to be classified for eye irritation (UN GHS: Category 2, H319).

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:

from 2017-11-21 to 2017-12-19

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol SkinEthic™ Skin Corrosivity Test

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number: 17-RHE-118
- Date of initiation of testing: 2017-11-21

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsing with a minimum volume of 20 mL DPBS using a pipette
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 15 min
- Spectrophotometer: microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm
- Filter: filter test plate

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%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 20 ± 3 mg per tissue

NEGATIVE CONTROL
- Amount applied: 40 ± 3 µL per tissue


POSITIVE CONTROL
- Amount applied: 40 ± 3 µL per tissue
- Concentration: 8N

Duration of treatment / exposure:

3 min or 1 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

104.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

87.7

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Summary of the results

Group		Tissue 1		Tissue 2		Mean		CV
		OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	3 min	1.849	98.4%	1.908	101.5%	1.879	100.0%	2.2%
	1 hour	1.820	99.7%	1.832	100.3%	1.826	100.0%	0.4%
Positive Control	1 hour	0.008	0.4%	0.011	0.6%	0.010	0.5%	20.0%
Test Item	3 min	1.884	100.3%	2.048	109.0%	1.966	104.7%	5.9%
	1 hour	1.550	84.9%	1.651	90.4%	1.601	87.7%	4.4%

Interpretation of results:

other: Not category 1 (corrosive) based on GHS criteria

Conclusions:

In an in vitro skin corrosion test (reconstructed human epidermis model, RHE), a cell viability of 104.7 % after 3 min incubation and 87.7 % after 1 h incubation was determined. According to the evaluation criteria, the test substance did not show skin corrosive properties.

Executive summary:

The corrosive potential of the test item was determined in a skin corrosion assay (RhE) according to OECD Guideline 431. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin corrosion potential. Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 40 ± 3 µL of either the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. Before application of 20 ± 3 mg of the solid test item, 20 ± 2 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. Positive and negative controls were valid. Following treatment with the test item the tissue viability was >50 % after 3 minutes exposure (mean viability: 104.7 %) and >15 % after 1 hour exposure (mean viability: 87.7 %). Thus, the test item is not considered as corrosive to skin.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 January 2018 - 22 March 2018

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:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number(s): 18-RHE-023
- Date of initiation of testing: On day of receipt the pre-incubation phase of the tissues started.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with minimum 25 mL DPBS; excess DPBS removed by shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Germany
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin category 2 if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg

NEGATIVE CONTROL
- Amount applied: 16 µL

POSITIVE CONTROL
- Amount applied: 16 µL
- Concentration: 5%

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

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

Run / experiment:

Tissue 2

Value:

104.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3

Value:

92.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not considered to possess an irritant potential to skin.

Executive summary:

The objective of the present study according to OECD guideline 439 was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 u,L of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 uL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met. Following treatment with the test item, the tissue viability was 92.5% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

9 January 2018 - 7 March 2018

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:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Age of donor animals: 18-80 months
- Corneal diameter: 24 - 27 mm
- Storage, temperature and transport conditions of ocular tissue: in transport medium cooled on ice
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL of the dissolved test item (i.e. 0.15 g/0.75 mL 0.9% sodium chloride solution)

VEHICLE
- Amount applied: 0.75 mL
- Concentration: 0.9%

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 corneas per group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded. Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. At the end of the incubation period, the basal opacity was determined with a calibrated opacitometer. The light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values). Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

NUMBER OF REPLICATES: 3

SOLVENT CONTROL USED: 0.9% sodium chloride solution

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL of the dissolved test item (i.e. 0.15 g/0.75 mL 0.9% sodium chloride solution) and 240 min

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE:
After the incubation period the corneal surface was washed three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The light transmission through the corneas was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany).
- Corneal permeability: The amount of fluorescein that crossed the cornea was measured spectrophotometrically with a microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- The following formula (referring to OECD Guideline 437) was used to determine the In Vitro Irritancy Score (IVIS) of the negative control:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
- The following formula was used to determine the In Vitro Irritancy Score (IVIS) of the positive control and the test item:
IVIS = corrected opacity value + (15 x corrected permeability OD490 value)
- The In Vitro Irritancy Score (IVIS) was calculated for each individual treatment and positive control cornea. The mean In Vitro Irritancy Score (IVIS) value of each treated group was calculated from the individual In Vitro Irritancy Score (IVIS) values.

DECISION CRITERIA:
IVIS ≤ 3 No Category (according to GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Serious eye damage according, Category 1 (according to GHS)

ACCEPTANCE CRITERIA:
A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean (IVIS positive control: 82.0 - 132.5).
The negative control responses should result in an IVIS that falls within three standard deviations of the current historical mean (IVIS negative control: -1.4 - 3.1).
A single test run with three corneas should be sufficient for a test item when the resulting classification is unequivocal. In cases of the following borderline results in the first testing run, a second test run should be considered.
- 2 of the 3 corneas give discordant predictions from the mean of all 3 corneas or
- 1 of the 3 corneas give discordant predictions from the mean of all 3 corneas, and the discordant result is >10 IVIS units from the cut-off threshold of 55

Irritation parameter:

in vitro irritation score

Remarks:

Replicate 1

Run / experiment:

1

Value:

24.09

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

Replicate 2

Run / experiment:

1

Value:

16.355

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

Replicate 3

Run / experiment:

1

Value:

15.08

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: Yes
- Acceptance criteria met for positive control: Yes

Interpretation of results:

other: not Category 1 (irreversible effects on the eye) based on GHS

Conclusions:

Based on the results of the present study, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (no Category).

Executive summary:

The objective of the present study according to OECD 437 was to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.5 (study acceptance criteria range: -1.4 - 3.1). Treatment with the positive control (20% Imidazole) revealed an IVIS of 129.6 (study acceptance criteria range: 82.0 - 32.5). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 18.5 and, thus higher than 3 and lower than 55. The test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (no Category).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

6 April 2018 - 5 June 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcular™ Tissues (OCL-200, OCL-212) (Lot No: 27036) was obtained from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia. Tissue funtionality and quality criteria were in the acceptance ranges and the QA statement reported that all criteria were passed.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg/tissue

For positive and negative control 50 µL were applied per tissue.

Duration of treatment / exposure:

6 +/- 0.25 h

Duration of post- treatment incubation (in vitro):

Directly after rinsing the tissues were incubated in assay medium for 25 min at room temperature. After the 25 min the tissues were transferred in fresh assay medium and incubated at 37 °C for 18 +/- 0.25 h.

Number of animals or in vitro replicates:

2 for test item, positive and negative control

Details on study design:

- Details of the test procedure used
The direct MTT reduction and coloring/staining properties of the test item were assessed before tissue treatment.
After shipment the tissues were removed from shipping containers and transferred into 6-well plates with fresh assay medium. Tissues were incubated overnight at 37 °C and 5 % CO2. Afterwards tissues were pre-wetted with 20 µL DPBS and incubated at 37 °C and 5 % CO2 for 30 minutes. Following the tissues were treated with test item or positive or negative control and incubated at 37 °C and 5 % CO2 for 6 hours (± 15 minutes). The treatment was stopped with rinsing the tissues with room temperature DPBS by submersion and gentle swirling for at least 3 times per beaker (3 beakers).Afterwards post-exposure immersion (25 min at room temperature) and post-exposure incubation (18 +/- 0.25 h at 37 °C and 5 % CO2) followed. After the post-treatment incubation period, the treated tissues were transferred in 300 µL MTT solution (1.0 mg/mL MTT) and incubated for 180 minutes (± 10 minutes) at 37 °C and 5 % CO2. Tissues were removed, blotted on absorbent material, and then transferred to a 6-well plate containing 2 mL isopropanol so that no isopropanol was flowing into the inserts. To extract the MTT, the plate was placed on an orbital plate shaker and shaken for 2-3 h at room temperature. The corresponding negative and positive controls were treated identically. The ODs of the extract solutions measured at 570 nm wavelength.
- RhCE tissue construct used, including batch number
EpiOcular™ Tissues (OCL-200, OCL-212) (Lot No: 27036) from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Doses of test chemical and control substances used
test item: 50 mg/tissue; positive and negative control 50 µL/tissue
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
treatement: 6 +/- 0.25 h; post-exposure immersion: 25 min at room temperature; post-exposure incubation: 18 +/- 0.25 h at 37 °C
- Number of tissue replicates used per test chemical and controls (positive control, negative control)
2 each
- Wavelength used for quantifying MTT formazan
570 nm ( Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Description of the method used to quantify MTT formazan
OD measurement
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Prediction model according to OECD 492 was used. The test item is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability is more than 60 %. In this case no further testing in other test methods is required. If the mean percent tissue viability is less than or equal 60 %, no prediction can be made. In this case, further testing with other test methods will be required because RhCE test methods show a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
negative control: OD >0.8 and <2.5
positive control: mean relative viability below 50 % of control viability (6 h exposure)
- Acceptable variability between tissue replicates for positive and negative controls
< 20 %
- Acceptable variability between tissue replicates for the test chemical
< 20 %

Irritation parameter:

other: Tissue viability [%]

Run / experiment:

Mean

Value:

1.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

29.6

Irritation parameter:

other: Tissue viability [%]

Run / experiment:

Tissue 1

Value:

1.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

97.9

Positive controls validity:

valid

Remarks:

25.3

Irritation parameter:

other: Tissue viability [%]

Run / experiment:

Tissue 2

Value:

1.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

102.1

Positive controls validity:

valid

Remarks:

33.9

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Difference in viability between duplicates was < 20 % for test item, negative and positive control.

Interpretation of results:

other: no prediction can be made regarding the eye hazard potential

Conclusions:

Under the conditions of the present study, the test item is not identified as not requiring classifictaion (UN GHS No Category). Based on this study the eye hazard potential of the test item cannot be predicted.

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model.

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 2.034 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 29.6 % (study acceptance criterion: <50 %). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 1.2 % and, thus, lower than 60 %. Therefore, according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

 

To determine the corrosion or irritation potential of the test item for the skin a stepwise weight of evidence approach was done. A skin corrosion assay (RhE) according to OECD Guideline 431 was conducted resulting in no classification in Category 1. Therefore, a skin irritation study (OECD 439) was conducted.

 

Studies

OECD 431 (reference 7.3.1 -1)

The corrosive potential of the test item was determined in a skin corrosion assay (RhE) according to OECD Guideline 431. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin corrosion potential. Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 40 ± 3 µL of either the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. Before application of 20 ± 3 mg of the solid test item, 20 ± 2 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. Positive and negative controls were valid. Following treatment with the test item the tissue viability was >50 % after 3 minutes exposure (mean viability: 104.7 %) and >15 % after 1 hour exposure (mean viability: 87.7 %). Thus, the test item is not considered as corrosive to skin.

 

OECD 439 (reference 7.3.1 -2)

The objective of the study according to OECD guideline 439 was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5 % aqueous solution of sodium dodecyl sulfate) were met. Following treatment with the test item, the tissue viability was 92.5 % and, thus, higher than 50 %, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

 

Conclusion

Using the weight of evidence approach the results of the two in vitro studies on skin corrosion (OEDC 431) and skin irritation (OECD 439) demonstrate that the test item is not considered as requiring classification for skin corrosion (UN GHS Category 1) and is considered to be non-irritant to skin (UN GHS: No Category). Therefore, the test item is determined not to require classification for skin irritation/corrosion.

 

Eye irritation

 

To determine the corrosion or irritation potential of the test substance for the eye a stepwise weight of evidence approach was done. An Bovine Corneal Opacity and Permeability Test according to OECD 437 was conducted resulting in “No prediction can be made” and therefore, the test item was neither required classification in Category 1 (UN GHS) nor required no classification (UN GHS: No Category). Therefore an eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 was conducted in accordance with the top down approach described in the Guidance on an integrated approach on testing and assessment (IATA) for serious eye damage and eye irritation (Series on testing and assessment No. 263; version 2017-07-20).

 

Studies 

OECD 437 (reference 7.3.2 -1)

The objective of the study according to OCD 437 was to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20 % (w/v) suspension in a 0.9 % sodium chloride solution. As negative control 0.9 % sodium chloride solution and as positive control 20 % (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 0.5 (study acceptance criteria range: -1.4 - 3.1). Treatment with the positive control (20 % Imidazole) revealed an IVIS of 129.6 (study acceptance criteria range: 82.0 - 132.5). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 18.5 and, thus higher than 3 and lower than 55, i.e. according to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.

 

OECD 492 (reference 7.3.2 -2)

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. After treatment with the negative control (sterile deionized water) the mean OD was 2.034 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 29.6 % (study acceptance criterion: <50 %). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 1.2 % and, thus, lower than 60 %. Therefore, according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

 

Conclusion

Based on the result from the Bovine Corneal Opacity and Permeability Test according to OECD 437 a categorization into Category 1 was excluded. A classification as No category according to UN GHS was not possible due to the result of “No predication can be made”. The second in vitro study, the eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492, also determined that no prediction regarding classification of the test item can be made. Thereby demonstrating that classification as “No category” is not indicated based on in vitro studies with the test item. Using the top down approach described in the Guidance on IATA for serious eye damage and eye irritation an overall conclusion for the test item can be reached. The weight of evidence with the two conducted studies determined that the test substance is not to be classified as UN GHS Category 1 and No Category. Therefore, the test item is considered to be eye irritating and classified into Category 2 (UN GHS).

Justification for classification or non-classification

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

The available data for skin irritation/corrosion are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is not considered to be classified for skin irritation/corrosion (UN GHS: no category) under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EC) No 2019/521.

The available data for eye irritation are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is considered to be classified for eye irritation (UN GHS: Category 2, H319) under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EC) No 2019/521.