α,α,α-trifluoro-m-toluidine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 December 2010 to 10 December 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted in accordance with OECD Guideline 431 "In Vitro Skin Corrosion: Human Skin Model Test". This study was performed in compliance with UK GLP standards (SI 1999/3106 as amended by SI 2004/0994)).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals No. 431 "In Vitro Skin Corrosion: Human Skin Model Test" (adopted 13 April 2004)

Deviations:

yes

Remarks:

The acceptance criteria for the negative control deviated from that outlined in the OECD TG.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-trifluoromethylaniline

IUPAC Name:

3-trifluoromethylaniline

Details on test material:

Identification: 3-trifluoromethylaniline
Description: Extremely pale yellow liquid
Batch Number: 101009
Purity: 99.66 %
Date Received: 25 October 2010
Storage Conditions: Room temperature in the dark

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Source: not stated, Batch number: 101009
- Expiration date of the lot/batch: not supplied
- Purity test date: not supplied

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at room temperature in the dark
- Stability under storage conditions: Not stated
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Not required as test substance is applied neat
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): Not Applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No pre treatment was conducted
- Preliminary purification step (if any): N/A
- Preparation of a nanomaterial dispersion (incl. dilution): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not stated

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Model Kit 0.38cm3
- Tissue batch number(s): Not stated
- Production date: Not stated
- Shipping date: Not stated
- Delivery date: 07 December 2010
- Date of initiation of testing:08 December 2010

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room Temperature
- Temperature of post-treatment incubation (if applicable): Room Temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Number of washing steps were not indicated. The samples were washed for 40 seconds
- Observable damage in the tissue due to washing: Not stated
- Modifications to validated SOP: Not stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: not stated
- Filter bandwidth: Not Stated
- Linear OD range of spectrophotometer: Not stated

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Not stated
- Barrier function: Not stated
- Morphology: Not stated
- Contamination: Not Stated
- Reproducibility: Not Stated

NUMBER OF REPLICATE TISSUES: Study reports use of Duplicate tissues

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues - Not stated
- Procedure used to prepare the killed tissues (if applicable): Not stated
- N. of replicates : Not stated
- Method of calculation used: Percentage tissue viability

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if after 240 minutes the relative mean tissue viability (% of negative control) is ≥ 35% .

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 ul of test material was applied
- Concentration (if solution): neat, 100%

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50ul
- Concentration (if solution): 0.9% sodium chloride solution

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50ul
- Concentration (if solution): Concentration of glacial acetic acid not stated

Duration of treatment / exposure:

3 minutes, 60 minutes, 240 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Duplicated tissues stated

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

205.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

122.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 minutes exposure

Value:

58.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not stated
- Direct-MTT reduction: the test item did not reduce MTT.
- Colour interference with MTT: the MTT solution containing the test item did not turn blue/purple.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD540 of the tissue replicates treated with sodium chloride solution was 0.231 which is within the acceptance criteria of ≥ 0.115 and ≤ 0.4.
- Acceptance criteria met for positive control: the relative mean tissue viability was 5.2% relative to the negative control treated tissues following the 240 minute exposure period which is within the acceptance criteria of 0 to 20% relative to the negative control following 240 minute exposure period
- Acceptance criteria met for variability between replicate measurements: Not stated
- Range of historical values if different from the ones specified in the test guideline: historical data not provided.

Any other information on results incl. tables

Table 1. Mean OD540 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	Exposure Period	Mean OD540 of Duplicate Tissues	Relative Mean Vaibility (%)
Negative Control Item	240 Minutes	0.231	100
Positive Control Item	240 Minutes	0.012	5.2
Test Item	240 Minutes	0.136	58.9
	60 Minutes	0.284	122.9
	3 Minutes	0.475	205.6

Applicant's summary and conclusion

Interpretation of results:

other: The test item was considered to be non corrosive to the skin according to OECD TG 431

Conclusions:

Under the conditions of the conducted test, the test substance did not possess corrosive properties towards reconstructed human epidermis tissue in the EpiSKIN model. No prediction on the skin irritation potential can be made and therefore additional testing should be conducted for classification and labelling purposes.

Executive summary:

the study was performed to OECD TG 431 under GLP conditions to assess the corrosive potential of the test material to Reconstructed Human Epidermis (RHE) following  single applciation. A volume of 50ul of the test item wsa applied topically to duplicate tissues ensuring uniform coverage of the tissues. duplicate tissues were also treated with 50ul of negative control 0.9% sodium chloride solution and positive control glacial acetic acid respectively. The RHE tissues samples were exposed to the test substance for 3, 60 and 240 minutes respectively with RHE tissues exposed to negative and positive controls for 240 minutes. Following the respective exposure times the tissues were rinsed for 40 seconds with PBD and incubated for 3 hours plus/minus 5 minues at room temperature. Under the conditions of this study, the test item was considered to not to be corrosive to the skin.