N,N'-methylenedistearamide

Endpoint summary

Description of key information

- The Reconstructed Human Epidermis test method performed according to OECD 439 under GLP, indicated that the test item was considered to be non-irritative to the skin.

- The Reconstructed Human Epidermis test method performed according to OECD 431 under GLP, indicated that the test item was considered to be non-corrosive to the skin.

- The Bovine Corneal Opacity and Permeability test (BCOP), performed according to OECD 437 under GLP, indicated that the test item is not considered to be irritating to the eye.

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:

21 Jun 2018 to 22 Jun 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Test system:

other: EpiDerm™ Reconstructed Human Epidermis Model Kit

Cell type:

non-transformed keratinocytes

Cell source:

other: Human-derived cells

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number: 28626
- Delivery date: 19 June 2018
- Date of initiation of testing: 21 June 2018

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader and LT-com analysis software
- Wavelength: 570 nm (OD570)
- Details on the method for assessing direct MTT reduction or colour interference can be found in 'Any other information on results incl. tables'.

METHODS
Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.
At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60-Minute positive control is < 15%.
- Reproducibility: In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

NUMBER OF REPLICATE TISSUES:
Duplicate tissues

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%.
- In addition, if test the test substance is considered to be corrosive based on the previous criteria, and if viability after 3 minutes exposure is smaller than 25%, the test substance is classified as sub-category 1A.
- In addition, if test the test substance is considered to be corrosive based on the previous criteria, and if viability after 3 minutes exposure is greater than or equal to 25%, the test substance is classified as a combination of sub-categories 1B and 1C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg of the test item (+ 25 μL of sterile water for wetting the test item)

NEGATIVE CONTROL: Sterile distilled water
- Amount applied: 50 μL

POSITIVE CONTROL: 8.0N Potassium Hydroxide
- Amount applied: 50 μL
- Concentration: 7.92M

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

3-Hour MTT incubation

Number of replicates:

Diplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean viability for 3-minute experiment

Value:

107.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean viability for 60-minute experiment

Value:

97.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.
- Colour interference with MTT: The solution containing the test item was a white colour. This colour was attributed to the intrinsic colour of the test item itself. It was therefore unnecessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.901 for the 3-Minute exposure period and 2.082 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Interpretation of results:

other: GHS criteria not met for corrosivity

Conclusions:

Based on this GLP compliant study performed according to OECD 431, the test item was considered to be non-corrosive to the skin.

Executive summary:

The skin corrosion potential of the test substance was evaluated in a GLP compliant study performed according to OECD 431, using the EpiDerm™ Reconstructed Human Epidermis Model Kit. In this model, corrosion is directly related to cytotoxicity, measured by the reduction of MTT to formazan by viable cells. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The negative and positive controls were valid and coefficient of variation did not exceed 30%. All acceptance criteria were therefore satisfied. The relative mean viability for the 3-minute exposure experiment was 107.3% and for the 60-minute exposure experiment 97.6%. Based on the classification criteria, the test item was considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

4 Jul 2018 to 9 Jul 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:

Updated 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, London Road, Shardlow, Derbyshire, DE72 2GD, UK,

Test system:

other: EPISKIN™ Reconstructed Human Epidermis Model Kit

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Human-derived cells

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number: 18-EKIN-027
- Delivery date: 3 Jul 2018
- Date of initiation of testing: 4 Jul 2018

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT solution
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader and LT-com analysis software
- Wavelength: 570 nm (OD570)
- Details on the method for assessing direct MTT reduction or colour interference can be found in 'Any other information on Materials and Methods incl. tables'.

METHODS
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 μL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant softstream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

NUMBER OF REPLICATE TISSUES: Triplicates

PREDICTION MODEL / DECISION CRITERIA
If the relative mean tissue viability is ≤50%, the substance is considered to be an irritant, but differentiation between EU CLP/UN GHS Category 1 and Category 2 will not be possible based on the results of this study.
If the relative mean tissue viability is >50%, the substance is not classified for irritation under EU CLP, but differentation between UN GHS non-irritant or Category 3 will not be possible based on the results of this study.

Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is 0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
Test Item: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg of the test item (+ 5 μL of sterile water for wetting the test item)

NEGATIVE CONTROL: DPBS
- Amount applied: 10 μL

POSITIVE CONTROL: SDS
- Amount applied: 10 μL
- Concentration: 5% w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

3-Hour MTT incubation

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean viability for 15-minute experiment

Value:

102.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.712 and the standard deviation value of the viability was 3.4%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 7.0% relative to the negative control treated tissues and the standard deviation value of the viability was 2.2%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 10.4%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

other: GHS criteria not met for skin irritation

Conclusions:

Based on this GLP compliant study performed according to OECD 439, the test item was considered to be non-irritative to the skin.

Executive summary:

The skin irritation potential of the test substance was evaluated in a GLP compliant study performed according to OECD 439, using the EpiSkin™ Reconstructed Human Epidermis Model Kit. In this model, irritation is directly related to cytotoxicity, measured by the reduction of MTT to formazan by viable cells. Triplicate tissues were treated with the test item for exposure periods of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The negative and positive controls were valid and coefficient of variation did not exceed 18%. All acceptance criteria were therefore satisfied. The relative mean viability for the 15-minute exposure experiment was 102.2%. Based on the classification criteria, the test item was considered to be non-irritative to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Jul 2018 to 24 Jul 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:

09 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir
- Characteristics of donor animals: typically 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue: The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics. They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- Time interval prior to initiating testing: within one day
- indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection and after incubation in Bovine Corneal Opacity and Permeability (BCOP) holders. Only corneas free of damage were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 μg/mL.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
For the purpose of this study the test item was used as supplied as the test item could not be formulated to a concentration of 20% w/v in sodium chloride 0.9% w/v as a suitable suspension/solution could not be obtained. An appropriately sized disc of the test item was applied to entirely cover the cornea and was found to weigh approximately 0.4900g.

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

A post-treatment opacity reading was performed immediately after removal of the test substance or controls. Followed an incubation time of 90 minutes for the permeability assay, as is described in details on study design.

Number of animals or in vitro replicates:

Triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 85 minutes. At the end of the incubation period each cornea was examined for defects.

QUALITY CHECK OF THE ISOLATED CORNEAS
Only corneas free of damage were used. The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken as reference point for each cornea, using a calibrated opacitometer.

NUMBER OF REPLICATES
Triplicates

NEGATIVE CONTROL USED
The negative control item, sodium chloride 0.9% w/v, was used as supplied.

POSITIVE CONTROL USED
The positive control item, Imidazole (purity >99%), was used as a 20% w/v solution in sodium chloride 0.9% w/v.

APPLICATION DOSE AND EXPOSURE TIME
For the purpose of this study the test item was used as supplied as the test item could not be formulated to a concentration of 20% w/v in sodium chloride 0.9% w/v as a suitable suspension/solution could not be obtained. An appropriately sized disc of the test item was applied to entirely cover the cornea and was found to weigh approximately 0.4900g.

TREATMENT METHOD: [closed chamber / open chamber]
The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the cornea. Approximately 0.4900 g of the solid test item was found to adequately cover the corneal surface. 0.75mL of each control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A post-treatment opacity reading was taken and each cornea was visually observed.
- Corneal permeability: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes. After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labelled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
- Others: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin. However, no histopathology was performed.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
The in vitro irritancy scores are summarized in Table 1 in 'Any other information on material and methods incl. tables'.

DECISION CRITERIA:
For an acceptable test the following positive control criterion should be achieved:
20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2017 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 71.2 to 132.9.

For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2017 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤2.3 and for permeability ≤0.044.

Irritation parameter:

in vitro irritation score

Run / experiment:

Based on #1, #2, #3

Value:

0.3

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Remarks:

corrected value

Run / experiment:

Mean #1, #2, #3

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein leakage

Remarks:

corrected value

Run / experiment:

Mean #1, #2, #3

Value:

0.023

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

CORNEAL EPITHELIUM CONDITION
The corneas treated with the test item were clear post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

CRITERIA FOR AN ACCEPTABLE TEST
The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤2.3 and permeability ≤0.044. The negative control acceptance criteria were therefore satisfied.

Individual and mean corneal opacity and permeability measurements can be found in Table 1 in 'Any other information on results incl. tables'.

 Table 1. Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD492)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	6	9	3		0.006
	2	4	4	0		0.000
	3	4	7	3		0.002
				2.0*		0.003##		2.0
Positive Control	4	5	72	67	65.0	1.075	1.072
	5	4	67	63	61.0	2.115	2.112
	6	3	62	59	57.0	1.430	1.427
					61.0#		1.537###	84.1
Test Item	7	6	8	2	0.0	0.051	0.048
	9	5	4	-1	0.0	0.011	0.008
	10	6	5	-1	0.0	0.014	0.011
					0.0#		0.023###	0.3

OD = Optical density

* Mean of the post-treatment

# Pre-treatment corrected values

## Mean permeability

## Mean corrected value

Interpretation of results:

GHS criteria not met

Conclusions:

This Bovine Corneal Opacity and Permeability test (BCOP), performed according to OECD 437 under GLP, indicated that the test item is not considered to be irritating to the eye.

Executive summary:

In this this eye irritation study performed according to OECD 437 under GLP, the irritative potential of the test substance was assessed ex vivo in a Bovine Corneal Opacity and Permeability test (BCOP). Bovine eyes were obtained from a local abattoir and the corneas were prepared and mounted in BCOP holders. Only corneas free of damage were used. Approximately 0.4900 g of the solid test item was found to adequately cover the corneal surface. For the negative control item (sodium chloride 0.9% w/v), and positive control item, (Imidazole (purity >99%)), 0.75 mL was applied to the corneas. The test was performed in triplicates. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes. After exposure, the corneas were washed 3 times with EMEM with phenol red, before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated.

The corneas treated with the test item were clear post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment. The in vitro irritancy scores are summarized as follows: test item 0.3, negative control 2.0, positive control 84.1. The controls were within the acceptable range, indicating the validity of the study. In conclusion, the test substance was not considered to be irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The skin irritation potential of the test substance was evaluated in a GLP compliant study performed according to OECD 439, using the EpiSkin™ Reconstructed Human Epidermis Model Kit. In this model, irritation is directly related to cytotoxicity, measured by the reduction of MTT to formazan by viable cells. Triplicate tissues were treated with the test item for exposure periods of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The negative and positive controls were valid and coefficient of variation did not exceed 18%. All acceptance criteria were therefore satisfied. The relative mean viability for the 15-minute exposure experiment was 102.2%. Based on the classification criteria, the test item was considered to be non-irritative to the skin.

Skin corrosion

The skin corrosion potential of the test substance was evaluated in a GLP compliant study performed according to OECD 431, using the EpiDerm™ Reconstructed Human Epidermis Model Kit. In this model, corrosion is directly related to cytotoxicity, measured by the reduction of MTT to formazan by viable cells. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The negative and positive controls were valid and coefficient of variation did not exceed 30%. All acceptance criteria were therefore satisfied. The relative mean viability for the 3-minute exposure experiment was 107.3% and for the 60-minute exposure experiment 97.6%. Based on the classification criteria, the test item was considered to be non-corrosive to the skin.

Eye irritation

In this this eye irritation study performed according to OECD 437 under GLP, the irritative potential of the test substance was assessed ex vivo in a Bovine Corneal Opacity and Permeability test (BCOP). Bovine eyes were obtained from a local abattoir and the corneas were prepared and mounted in BCOP holders. Only corneas free of damage were used. Approximately 0.4900 g of the solid test item was found to adequately cover the corneal surface. For the negative control item (sodium chloride 0.9% w/v), and positive control item, (Imidazole (purity >99%)), 0.75 mL was applied to the corneas. The test was performed in triplicates. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes. After exposure, the corneas were washed 3 times with EMEM with phenol red, before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visuallyobserved. Followingthe opacity measurement the permeability of the corneas to sodium fluorescein was evaluated.

The corneas treated with the test item were clear post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment. The in vitroirritancy scores are summarized as follows: test item 0.3, negative control 2.0, positive control 84.1. The controls were within the acceptable range, indicating the validity of the study. In conclusion, the test substance was not considered to be irritating to the eye.

Justification for classification or non-classification

Based on the available information, classification for skin and eye irritation is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.