4,4'-bis(chloromethyl)-1,1'-biphenyl

Endpoint summary

Description of key information

Eye Irritation (in vitro)

Under the conditions of the study, the test material is not considered an irritant.

Skin Corrosion (in vitro)

Under the conditions of this study, the test material was not corrosive to the skin.

Skin Irritation (in vitro)

Under the conditions of the study, the results for the in vitro EPISKIN model test with the test material indicates that the test material is non-irritant to skin.

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:

key study

Study period:

8 December 2016 to 9 December 2016

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: EU Method B.40.Bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test material was applied as supplied, no formulation was required (although the test material was ground to a fine powder).
No correction for purity of the test material was applied.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN™(SM) model has been validated for corrosivity testing in an international trial and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™(SM) three-dimensional human epidermis model
- Source: SkinEthic
- Tissue lot number(s): 16-EKIN-049
- Expiry date: 12 December 2016
- The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells.

TEST FOR DIRECT MTT REDUCTION
20 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a > 95% humidified atmosphere for 3 hours and then any colour change was observed.
If the MTT solution containing the test material turned blue/purple relative to the control, the test material was presumed to have reduced the MTT.

ASSESSMENT OF COLOURING POTENTIAL OF TEST MATERIAL
Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol. As the test material had an intrinsic colour, further evaluation was necessary to detect colouring potential. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.

MAIN TEST
PRE-INCUBATION
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2 in a > 95% humidified atmosphere.

APPLICATION
The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- 20 mg of test material was applied evenly to the epidermal surface of each of two test units and then 100 μL physiological saline was added to the test material to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.
The plates with the treated epidermis units were incubated for 4 hours (± 10 min) at room temperature (22.9 - 24.2°C) covered with the plate lids.

NUMBER OF REPLICATE TISSUES:
- Test material: 2
- Negative controls: 2
- Positive controls: 2
Furthermore, as the test item was coloured, two additional test material-treated tissues were used for the non specific OD evaluation.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 22.9 - 24.2°C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the incubation time (4 hours), all test material treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly. The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MTT TEST
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except for the two living colour control units). The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours (± 15 min), protected from light.

FORMAZAN EXTRACTION
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 2×200 μL samples from each tube were placed into the wells of a labelled 96-well plate. The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate at the required wavelength on each day before use.

DATA EVALUATION
- The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD570 of the test material / mean OD570 of negative control) x 100

INTERPRETATION OF RESULTS
If both disks have mean viability of ≥ 35% = Non Corrosive
If both disks have mean viability of < 35% = Corrosive (at the corresponding incubation period)

Otherwise:
If the mean value is ≥35% and the variability is less than 50% = Non Corrosive
If the mean value is <35% and the variability is less than 50% = Corrosive
Otherwise:
If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35%. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.

VALIDITY OF THE TEST
The mean OD value of the two negative control tissues should be ≥ 0.6 and ≤ 1.5 and negative control OD values should not be below historically established boundaries.
The acceptable mean viability % range for positive controls is ≤ 20%.
The difference of viability between the two tissue replicates should not exceed 30%.
The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): 0.9% (w/v) NaCl solution

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of post-treatment incubation (if applicable):

4 hours

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test material

Value:

92

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

positive control

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION
The MTT solution containing the test material did not turn blue/purple. This was taken to indicate the test material did not reduce MTT.

ADDITIONAL CONTROLS
As the test material was coloured, two additional test material-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.002, Non Specific Colour % was calculated as 0.3%. This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

VIABILITY RESULTS
The mean OD value for the test material treated skin samples showed a 92.0% relative viability.
The mean OD value for the positive control treated skin samples showed a 0.8% relative viability.

VALIDITY OF THE TEST
- After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper condition.
- The mean OD value of the two negative control tissues was in the recommended range (0.836).
- The two positive control treated tissues showed 0.8% viability demonstrating the proper performance of the assay.
- The difference of viability between the two test material-treated tissue samples in the MTT assay was 13.9%.
- The difference of viability between the two negative control tissue samples in the MTT assay was 15.0%.
- The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical density (OD)			Viability (%)
	Measured		Blank corrected
Negative control	1	0.819	0.773	92.5
	2	0.944	0.898	107.5
	mean	-	0.836	100.0
Positive control	1	0.052	0.006	0.8
	2	0.053	0.007	0.8
	mean	-	0.007	0.8
Test material	1	0.868	0.822	98.4
	2	0.761	0.715	85.5
	mean	-	0.768	92.0

 

Interpretation of results:

other: Not corrosive according to EU criteria

Conclusions:

Under the conditions of this study, the test material was not corrosive to the skin.

Executive summary:

The skin corrosion potential of the test material was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40.bis, under GLP conditions.

During the study, disks of EPISKIN™(SM) were treated with test material and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution from the test material. For each treated tissue viability was expressed as a % relative to the negative control.

Following exposure with the test material, the mean cell viability was 92.0% compared to the negative control. This is above the threshold of 35%, therefore the test material was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test, the results indicate that the test item is non corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 February 2017 to 25 February 2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was applied as supplied, no formulation was required (although it was grounded to fine powder).

Test system:

human skin model

Remarks:

EPISKIN™ (SM) reconstituted human epidermis (three-dimensional human epidermis model)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult human-derived epidermal keratinocytes

Source strain:

other: Not applicable

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: SkinEthic, France.
- Model: EPISKIN™ (SM) (0.38 cm2)

Justification for test system used:

The EPISKIN™ (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ (SM). The EPISKIN-SM is three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
EPISKIN™ (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
- Tissue batch number(s): Batch No.:17-EKIN-008
- Expiry date: 27 February 2017
- Date of initiation of testing: 22 February 2017
- Quality control: EPISKIN™ (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKIN™ (SM) test kits used in the present study)
- Kit Reception: The pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40 °C (the colour change is irreversible, independent of the length of the period above 40 °C):
-white colour = good
-grey or black colour = not acceptable
The kits were found to be in good order at reception.
- Storage: The EPISKIN™ (SM) kit was kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.
Procedures described below were performed under aseptic conditions (in sterile hood using sterile equipment).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (25.7-26.3 °C).
- Temperature of post-treatment incubation: 37 °C
Pre-incubation (Day [-1])
The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37 °C in an incubator with 5 % CO2, in a >95 % humidified atmosphere.
As the test item was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 10 mg of the powdered test item was applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After incubation, the EPISKIN™ (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette without touching the epidermis.
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37 °C in an incubator with 5 % CO2 in a >95 % humidified atmosphere.
- Observable damage in the tissue due to washing: None specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well.
- Incubation time: 3 hours ± 5 min.
- Spectrophotometer: Not specified
MTT solution
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 21 February 2017) was stored in refrigerator (2-8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

Acidified Isopropanol
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

INDICATOR FOR POTENTIAL FALSE VIABILITY
Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement.

MTT test (Day 2)
After the 42 hours incubation, all EPISKIN™ (SM) units (except of two colour control units) were transferred into the MTT working solution filled wells. Then, all transferred EPISKIN™ (SM) units were incubated for 3 hours (± 5 min) at 37 °C in an incubator with 5 % CO2 in a >95 % RH% protected from light.

Formazan extraction (Day 2)
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.


NUMBER OF REPLICATE TISSUES: In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation.

Cell viability measurements (Day 2)
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
10 mg of powdered test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in a shaking water bath for 3 hours (±5 minutes) protected from light, and then any colour change was recorded:
-Test items which do not react with MTT: Yellow
-Test items reacting with MTT: bBue or purple
After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.

Check-method to detect the colouring potential of test-items:
Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water* and/or extracting solution (e.g. acidified isopropanol) (simulating a tissue humid environment). As the test item had an intrinsic colour, thus further evaluation to detect colouring potential was necessary.
Non-Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues.
*Note: Water is the environment during exposure.
Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non-specific OD evaluation. This tissue followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

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

PREDICTION MODEL / DECISION CRITERIA
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is less or equal (≤) to 50 % of the mean viability of the negative controls. In case the test chemical is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2. The test item considered to be non-irritant to skin (No Category), if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than (˃) to 50 % of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: As the test item was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 10 mg of the powdered test item was applied evenly to the epidermal surface.

NEGATIVE CONTROL
- Amount applied: 50 μL of negative control Phosphate Buffered Saline (PBS) was added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).

POSITIVE CONTROL
- Amount applied: The positive control 5 % (w/v) Sodium Dodecyl Sulphate solution was added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
- Preparation: The positive control solution was prepared freshly in the testing laboratory.

Duration of treatment / exposure:

Disks of EPISKIN™ (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS).

Duration of post-treatment incubation (if applicable):

The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2 in a >95 % RH %.

Number of replicates:

In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of results

Value:

63.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not specified
- Direct-MTT reduction: As no colour change (yellow colour) was observed after three hours of incubation of the test items in MTT working solution, thus the test materials did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
- Colour interference: As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.013, Non Specific Colour % was calculated as 1.8 %. This value was below 5 %, therefore additional data calculation was not necessary.

ACCEPTANCE OF RESULTS:
The OD values for the test item treated skin samples showed 63.3 % relative viability.
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was in the recommended range (0.681). Standard deviation of the viability results for negative control samples was 2.2.
- Acceptance criteria met for positive control: The positive control treated tissues showed 5.8 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.9.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 2.2. The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study the test material is non-irritant to skin.

Executive summary:

A study was performed in vitro to assess the irritancy potential of the test material in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

Disks of EPISKIN™ (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2 in a >95 % RH %. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2 in a >95RH % protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSC living) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control, the test item is considered to be irritant to skin.

Following exposure with the test material, the mean cell viability was 63.3 % compared to the negative control. This is above the threshold of 50 %, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Under the conditions of the study the test material is non-irritant to 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:

07 March 2016 to 07 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Provided by sponsor
- Batch/Lot Number: 150701
- Expiration date of the lot/batch: 14 July 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25 ˚C), protected from humidity, under inert gas

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Number of animals: Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.
- Characteristics of donor animals: Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.
- indication of any existing defects or lesions in ocular tissue samples: One small drop of 2 % (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg of the powdered test item.

VEHICLE
The test item was applied as supplied. No formulation was required; although the test item was powdered.

Duration of treatment / exposure:

10 seconds from the end of the application to the cornea surface.

Duration of post- treatment incubation (in vitro):

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

Number of animals or in vitro replicates:

3 corneas were exposed to the test material. Three positive control treated eyes and one negative control eye were examined during the study.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. Fluorescein was used to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
Preparation of eyes
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5 °C) during the acclimatization and treatment periods.
The baseline assessments
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5 % within the -45 min and the zero time. No changes in thickness (0.0 %) were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
Three

NEGATIVE CONTROL USED
30 µL physiological saline (0.9 % (w/v) NaCl).

SOLVENT CONTROL USED (if applicable)
Not applicable. The test item was applied as supplied. No formulation was required; although the test item was powdered.

POSITIVE CONTROL USED
30 mg of powdered Imidazole.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test item was applied onto the centre of the cornea. For treatment, 30 mg of the powdered test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly.

OBSERVATION PERIOD
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item if possible.
Additional gentle rinsing with 20 mL saline was performed at each time point when the control material remaining on the cornea was observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
- Damage to epithelium based on fluorescein retention: Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Corneal thickness was measured at all time points using a Haag-Streit Bern 900 slit-lamp microscope

SCORING SYSTEM:
- Mean corneal swelling (%)
Corneal swelling was calculated according to the following formulae:

CS at time t =( (CT at time t-CT at t=0) / (CT at t = 0)) x 100
Mean CS at time t = (FECS(at time t) + SECS(at time t) + TECS(at time t)) / 3

Where:
CS = cornea swelling
CT = cornea thickness
FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point
Small negative numbers for swelling (0 to - 5 %) following application are evaluated as class I. Large negative numbers (>12 % below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 % to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

- Mean maximum opacity score
Cornea opacity was calculated according to the following formulae:

ΔCO at time t = CO at time t – CO at t = 0

Mean ΔCOmax = (FECO(30 min to 240 min) + SECO(30 min to 240 min) + TECO(30 min to 240 min)) / 3

Where:
CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
CO at t=0 = baseline cornea opacity
ΔCO at time t = difference between cornea opacity at t time and cornea opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

- Mean fluorescein retention score at 30 minutes post-treatment
Fluorescein retention was calculated according to the following formulae:

ΔFR at time t = FR at time t –FR at t = 0
Mean FRΔ = (FEFR(30 min) + SEFR(30 min) + TEFR(30 min)) / 3

Where:
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
ΔFR at time t = difference between fluorescein retention at t time and fluorescein retention baseline
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention

DECISION CRITERIA: The conclusion on eye irritancy was based on the OECD guideline quantitative assessments.

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Remarks:

It is concluded that further information is required for classification.

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Remarks:

It is concluded that further information is required for classification.

Irritation parameter:

percent corneal swelling

Remarks:

at up to 240 min

Run / experiment:

Mean

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: -1.6 % It is concluded that further information is required for classification.

Remarks:

Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Irritation parameter:

other: Overall ICE Class

Run / experiment:

Mean

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 3xI

Other effects / acceptance of results:

OTHER EFFECTS:
Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Based on these in vitro eye irritation assays in isolated chicken eyes, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control, physiological saline, was classified as non-irritating.
- Acceptance criteria met for positive control: The positive control, Imidazole, was classified as severely irritating; UN GHS Classification: Category 1.

Validity of the test

The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

Test item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	-0.5 %	I
Mean maximum corneal swelling at up to 240 min	-1.6 %	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.0	I
Other Observations	Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	3xI

The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention ICE classes are used for classification.

 

Positive control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	8.1 %	II
Mean maximum corneal swelling at up to 240 min	25.8 %	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII; 2xIV

The positive control (Imidazole) was classified as severely irritating, UN GHS Classification: Category 1.

 

Negative control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0 %	I
Mean maximum corneal swelling at up to 240 min	0.0 %	I
Mean maximum corneal opacity	0.0 %	I
Mean fluorescein retention	0.0 %	I
Other Observations	None
Overall ICE Class	3 x I

The negative control Physiological saline was classified as non-irritating, UN GHS Classification: Non-classified.

 

Historical Control data (on 24 September 2015): Physiological saline

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-3.2 %	3.4 %
Maximum corneal swelling at up to 240 min	-4.8 %	3.4 %
Maximum corneal opacity change	0.00	0.50
Fluorescein retention	0.00	0.50
Number of studies	200

 

Historical Control data (on 24 September 2015): Imidazole

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-6.6 %	25.0 %
Maximum corneal swelling at up to 240 min	-15.9 %	35.9 %
Maximum corneal opacity change	3.50	4.00
Fluorescein retention	2.00	3.00
Number of studies	96

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the study, the test material is not considered an irritant. Further information is required for classification.

Executive summary:

The eye irritancy potential of the test material was investigated in vitro in a study conducted in accordance with the standardised guidelines OECD 438 and EU Method B.48 under GLP conditions.

30 mg powdered test material was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % (w/v) NaCl solution). In the study, three test material treated eyes, three positive control treated eyes and one negative control treated eye were examined. The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the experiment was considered to be valid. No significant corneal swelling and no corneal opacity change were observed during the four hour observation period. No fluorescein retention change was noted on all three eyes. However, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. This fact might indicate morphological changes in an in vivo system (although during in vivo conditions the eyelids will probably clear the surface, but abrasion may occur).

Under the conditions of the study, the test material is not considered an irritant. However, It is concluded that further information is required for classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Eye Irritation (in vitro)

The eye irritancy potential of the test material was investigated in vitro in a study conducted in accordance with the standardised guidelines OECD 438 and EU Method B.48 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

30 mg powdered test material was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % (w/v) NaCl solution). In the study, three test material treated eyes, three positive control treated eyes and one negative control treated eye were examined. The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the experiment was considered to be valid. No significant corneal swelling and no corneal opacity change were observed during the four hour observation period. No fluorescein retention change was noted on all three eyes. However, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. This fact might indicate morphological changes in an in vivo system (although during in vivo conditions the eyelids will probably clear the surface, but abrasion may occur).

Under the conditions of the study, the test material is not considered an irritant.

Skin corrosion (in vitro)

The skin corrosion potential of the test material was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40.bis, under GLP conditions.

During the study, disks of EPISKIN™(SM) were treated with test material and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution from the test material. For each treated tissue viability was expressed as a % relative to the negative control.

Following exposure with the test material, the mean cell viability was 92.0% compared to the negative control. This is above the threshold of 35%, therefore the test material was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test, the results indicate that the test item is non corrosive to the skin.

Skin Irritation (in vitro)

A study was performed in vitro to assess the irritancy potential of the test material in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

Disks of EPISKIN™(SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2 in a >95 % RH %. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2 in a >95RH % protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSC living) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control, the test item is considered to be irritant to skin.

Following exposure with the test material, the mean cell viability was 63.3 % compared to the negative control. This is above the threshold of 50 %, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Under the conditions of the study the test material is non-irritant to skin.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to eye irritation and skin irritation.