3',4',5'-trifluorobiphenyl-2-amine

Endpoint summary

Description of key information

OECD TG 439: not irritating to skin

OECD TG 492: irritating to eye

HET-CAM Test: no serious eye damage to be expected

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: SK03/186

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature; no direct sunlight

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

other: PBS

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall
and for 35 minutes in the incubator.

REMOVAL OF TEST MATERIAL AND CONTROLS
The tissues were washed with sterile PBS 1 hour after start of application.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

DECISION CRITERIA
Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

25 μL sterile PBS was applied first. Thereafter, a bulk volume of ca. 25 μL of the solid test material was applied and homogeneously distributed together with the fluid.

Control tissues were concurrently applied with 30 μL of sterile PBS (NC) or with 30 μL of 5% SDS (PC).

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours
After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

93.3

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

101.7

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

101.7

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

98.9

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Conclusions:

Based on the observed results and applying the evaluation criteria, it was concluded, that [1,1'-Biphenyl]-2-amine, 3',4',5'-trifluoro- does not show a skin
irritation potential in the EpiDerm™ in vitro skin irritation test under the test conditions chosen.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: SK03/186

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature; no direct sunlight

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium

Assay medium: OCL-200-ASY assay medium
MTT diluent: Dulbecco's modified eagle's medium (DMEM) based medium used for diluting MTT
Pre-treatment / wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+
Detection agent: 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT), 1.0 mg / mL MTT diluent
Extracting agent: Isopropanol p.a.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Using a sharp spoon, a bulk volume of ca. 50 μL of the test material was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC).

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

EXPERIMENTAL PROCEDURE

To assess the ability of the test material to directly reduce MTT a pretest was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently. If the color of the MTT
solution or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
-Basic procedure:
Two tissues were treated with each, the test substance, the PC and the NC.
Pre-incubation of the tissues:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.
-Pretreatment of the tissues:
After the pre-incubation the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
-Application of the test substance:
A bulk volume of ca. 50 μL of the test material was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC).
After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.
-Removal of the test substance and postincubation period:
To remove the test substance, the tissues were washed with sterile PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium in order to remove residual test substance. After 25 minutes of post-soak immersion, each tissue was dried on absorbent paper and
transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 18 hours
-MTT incubation:
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation.
The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

- RhCE tissue construct used, including batch number:
Tissue model: OCL-200
Tissue Lot Number: 21593
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- Evaluation of results:
The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 60%.

Irritation parameter:

other: viability

Run / experiment:

1

Value:

29.7

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: viability

Run / experiment:

2

Value:

26.7

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: viability

Run / experiment:

mean

Value:

28.2

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: no

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the observed results and applying the evaluation criteria it was concluded, that [1,1'-Biphenyl]-2-amine, 3',4',5'-trifluoro- shows an eye irritation potential in the EpiOcular™ in vitro eye irritation test under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data available and according to Regulation (EC) 1272/2008 the test substance fulfills the criteria for classification with H319 (causes serious eye irritation).