Biphenyl-4-yl-(9,9-dimethyl-9H-fluoren-2-yl)-[2-(9,9-diphenyl-9H-fluoren-4-yl)-phenyl]-amine

Endpoint summary

Description of key information

According to the OECD Guiedeline 431 the test item is not considered to possess a corrosive potential to skin.

Accroding to the OECD Guideline 439 the test item was not irritating to skin.

According to the OECD Guideline 437 the test item did not show any eye hazard potential.

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:

25 January 2017 - 25 January 2017

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:

July 29, 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

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol SkinEthic™ Skin Corrosivity Test

Version / remarks:

April 2012

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Episkin/SkinEthic Laboratories, Lyon, France

Source strain:

not specified

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(s): 17-RHE-009
- Date of initiation of testing: 25 January 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 mL DPBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 hours (+/- 15 minutes)
- Spectrophotometer:ELx800, BioTek Instruments GmbH,
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 1.4 (Acceptance criteria: OD > 0.7)
- Barrier function: 5.5 h (Acceptance criteria: 4 h <= ET50 <= 10 h
- Morphology: 5.5 cell layers, absent of significant histological abnormalities, satisfactory (acceptance criteria: number of cell layers >= 4; absence of significant histological abnormalities; well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum)

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates: one

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

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 20 mg (+/- 3 mg)

NEGATIVE CONTROL (deionised water)
- Amount applied: 40 ± 3 μL

POSITIVE CONTROL
- Amount applied: 40 ± 3 μL

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1/ 3 min

Value:

99.54

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1/ 1 hours

Value:

92.52

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2/ 3 min

Value:

112.98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2/ 1 hour

Value:

98.48

Vehicle controls validity:

not applicable

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:

Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin corrosion 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 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. After treatment with the negative control (deionized water) the mean OD per tissue replicate was 1.696 and 1.913 after 3 minutes exposure and 1.935 and 2. 145 after 1 hour exposure (study acceptance criterion: >= 0.8 and <= 3.0). Treatment with the positive control (Potassium hydroxide, 8N) revealed a mean viability of 0.48% after 1 hour (study acceptance criterion: <15%). Therefore, the study fulfilled the validity criteria. Following treatment with the test item, the tissue viability was >= 50% after 3 minutes exposure (mean viability: 106.26%) and >= 15% after 1 hour exposure (mean viability: 95.50%), i.e. according to OECD 431 the test item is not considered as corrosive to skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 28, 2018 - May 7, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No. 640/2012 amending, for the purpose of its adaption to technical progress, Regulations (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was applied neat to the tissues.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

standard model

Vehicle:

unchanged (no vehicle)

Remarks:

No vehicle used in this study; The test item was applied neat to the tissues.

Details on test system:

CELL CULTURE
- Supplier: EpiSkin/SkinEthic Laboratories, Lyon, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate4137
- Expires: April 23, 2018

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

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL: 16 mg ± 2 mg per tissue
NEGATIVE CONTROL: 16 µL (Dulbecco`s Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (5% aqueous solution of sodium dodecyl sulfate in deionised water)

Duration of treatment / exposure:

42 min (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1

Value:

105.6

Vehicle controls validity:

not applicable

Remarks:

The test item was applied neat to the tissues

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: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

Acceptance Criterion Result
Negative control OD ≥ 0.8 and ≤ 3.0 1.912 to 2.034

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

Acceptance Criterion Result
Mean OD negative control ≥ 1.2 1.953
Mean viability positive control < 40% 1.4%
SD of group-mean value ≤ 18% 14.3% (positive control)
3.6% (negative control)

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

Acceptance Criterion Result
Mean OD negative control ≥ 1.455 1.953
Mean viability positive control ≤ 2.97% 1.4%

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18% 3.3%

The study met all acceptance criteria.

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	1.953	100
Positive Control	42	0.028	1.4
Test Material	42	2.063	105.6

Interpretation of results:

GHS criteria not met

Conclusions:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

Executive summary:

Objective

The objective of the present study was to investigate the potential of the test item to induce skin irritation in anin vitrohuman skin model.

Study Design

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.

Results

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 105.6% 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

Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

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:

10 February 2017 - 10 February 2017

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:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: OdenwaldschlachthofBrensbach, 64395 Brensbach, Germany
- Characteristics of donor animals: 21 - 191 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were kept and transported 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: Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL of the suspended test item (i.e. 150 mg/750 µL)
- Concentration: 20% (w/v) suspension in a 0.9% sodium chloride solution

VEHICLE
- Amount applied: 750 µL

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice. The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. 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 (CiToxLAB, Veszprem, Hungary) 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
For equilibration, the corneas in the holder were incubated (BSS 160, Grumbach Brutgeräte GmbH, Asslar, Germany) in a vertical position at 32 ± 1°C for about one hour. At the end of the incubation period, the incubation medium was replaced by fresh pre-warmed
(32 ± 1°C) incubation medium in both compartments. The baseline opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). 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. Three corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control group.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: No

SOLVENT CONTROL USED: 0.9% sodium chloride solution

POSITIVE CONTROL USED : Imidazole

APPLICATION DOSE AND EXPOSURE TIME : 750 µL of the suspended test item (i.e. 150 mg/750 µL) and 240 min

TREATMENT METHOD: closed chamber (solvent and positive control); open chamber (test item)

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: To remove the test item from the epithelium, the window locking-ring and the glass window from the anterior chamber were removed. The corneas were gently rinsed with wash medium using a syringe. Before measurement of the opacity value after treatment, fresh incubation medium was replaced in both compartments. To remove thesolvent and positive control, the corneal surface was washed three times.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others: Each cornea was observed visually and pertinent observations were recorded (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: IVIS <= 3 (no UN GHS category), IVIS > 3; <= 55 (No prediction can be made), IVIS > 55 (UN GHS category 1)

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 1

Value:

0.315

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 2

Value:

1.815

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 3

Value:

3.385

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

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

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Executive summary:

The objective of the present study was to exarnine the potential of the test itern to induce serious eye darnage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro rnodel for ocular hazard assessrnent.

To deterrnine the eye hazard potential the induced opacity and increased perrneability was investigated in isolated bovine corneas after exposure to the test itern as a 20% (w/v) suspension in a 0.9% sodiurn chloride solution. As negative control 0.9% sodiurn chloride solution and as positive control 20% (w/v) lmidazole was used. Three corneas were used per group (negative control, positive control ortest itern group). The incubation time was 240 min. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 rninutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro lrritancy Score (IVIS). The study fulfilled the acceptance criteria.

The IVIS obtained after treatrnent with the test item was 1.8 and, thus, lower than 3, i.e. according to OECD 437 the test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The objective of the present study 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.

Following treatment with the test item, the tissue viability was 105.6% 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).

Skin corrosion

The objective of the present study was to investigate the potential of the test item to induce skin corrosion 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 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. After treatment with the negative control (deionized water) the mean OD per tissue replicate was 1.696 and 1.913 after 3 minutes exposure and 1.935 and 2. 145 after 1 hour exposure (study acceptance criterion: >= 0.8 and <= 3.0). Treatment with the positive control (Potassium hydroxide, 8N) revealed a mean viability of 0.48% after 1 hour (study acceptance criterion: <15%). Therefore, the study fulfilled the validity criteria. Following treatment with the test item, the tissue viability was >= 50% after 3 minutes exposure (mean viability: 106.26%) and >= 15% after 1 hour exposure (mean viability: 95.50%), i.e. according to OECD 431 the test item is not considered as corrosive to skin.

Eye irritation

The objective of the present study was to exarnine the potential of the test itern to induce serious eye darnage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro rnodel for ocular hazard assessrnent.

To deterrnine the eye hazard potential the induced opacity and increased perrneability was investigated in isolated bovine corneas after exposure to the test itern as a 20% (w/v) suspension in a 0.9% sodiurn chloride solution. As negative control 0.9% sodiurn chloride solution and as positive control 20% (w/v) lmidazole was used. Three corneas were used per group (negative control, positive control ortest itern group). The incubation time was 240 min. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 rninutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro lrritancy Score (IVIS). The study fulfilled the acceptance criteria.

The IVIS obtained after treatrnent with the test item was 1.8 and, thus, lower than 3, i.e. according to OECD 437 the test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Justification for classification or non-classification

Based on the availbale data, the test item is not classified and labelled for skin and eye irritation according to Regulation (EC) No 1272/2008.