Alkali metal [bis(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluoro-2- substitutednonyl)amino]alkanesulfonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 14th to 17th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4 - 45 °C

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Human adult donors

Justification for test system used:

The EPISKIN Small ModeITM (EPISKIN-SMTM) is one of the biological systems of the validated alternative method for skin irritation (OECD TG 439).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModeITM (EPISKIN-SMTM) supplied by EPISKIN Laboratoires, 4 Rue Alexander Fleming 69366 Lyon, France.
- Tissue batch number(s): N° 17-EKIN-007
- Delivery date: 14.02.2017
- Date of initiation of testing: 14.02.2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: washing were performed with saline solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: 42 hours
- Spectrophotometer: TECAN INFINITE M-200 spectrophotometer.
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 0-2 OD

NUMBER OF REPLICATE TISSUES: test item, positive and negative controls were evaluated in triplicate on viable episkin tissues.

INSTRUMENT USED FOR THE TEST:
Analytical bilance XS204 I
CO2 incubator
Microplate Autoreader INFINITE M200
Micropipettes
Refrigerator FRL 360

PREDICTION MODEL / DECISION CRITERIA
- If the mean relative tissue viability (%) of treated tissues exposed to the test substance is > 50 % the test substance is identified as not requiring classification and labeling(NO Category- Non-irritant to skin).
- If the mean relative viability is ≤ 50 % the test substance is identified as potentially requiring classification and labeling as Category 1 (risk phrase H314: causes severe skin burns and eye damage) or Category 2 (risk phrase H315: causes skin irritation).
In absence of information on skin corrosion the test method cannot resolve between UN GHS Categories 1 and 2.

ACCEPTANCE CRITERIA
- Acceptance criteria for negative control: negative control meets the acceptance if the mean OD negative control value of the three tissues is ≥ 0.6 and ≤ 1.5 and if the Standard Deviation value (SD) of the % viability is ≤ 18 %.
- Acceptance criteria for positive control: positive control satisfies acceptance criteria if mean cell viability expressed as percentage compared to Negative Control is ≤ 40 % and Standard Deviation value (SD) is ≤ 18 %.
- Acceptance criteria for test substance: test substance meets the acceptance criteria if the Standard Deviation value (SD) of the cell viability % (expressed as percentage compared to Negative Control) between the 3 tissue replicates is ≤ 18 %.

Control samples:

other: Positive (Sodium Dodecyl Sulfate at 5%) and negative (NaCl 0.9%) controls

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg of test item were directly and uniformly applied topically on epidermal surface pre-treated with 10 µL of saline solution.

NEGATIVE CONTROL
- Concentration (if solution):10 µL of saline solution (NaCl 0.9%) were directly and uniformly applied topically on epidermal surface.

POSITIVE CONTROL
- Concentration (if solution):10 µL of Sodium Dodecyl Sulfate at 5% (SDS 5%) were directly and uniformly applied topically on epidermal surface.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Test item, positive and negative controls were evaluated in triplicate on viable episkin tissues.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item mean of three replicates

Value:

ca. 104.16

Vehicle controls validity:

not valid

Remarks:

no vehicle used

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: no direct MTT reduction observed for test item in the qualitative visual inspection.
- Colour interference with MTT: no colour intereference with MTT observed for test item in the qualitative visual inspection.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: negative control meets the acceptance if the mean OD negative control value of the three tissues is ≥ 0.6 and ≤ 1.5 and if the Standard Deviation value (SD) of the % viability is ≤ 18 %.The mean OD negative control value of the three tissues was 0.87 (inside the range of acceptability 0.6 - 1.5) and the Standard Deviation value (SD) of the % viability was 2.87 (≤ 18% as indicated in the accepability criteria).
- Acceptance criteria met for positive control: positive control satisfies acceptance criteria if mean cell viability expressed as percentage compared to negative control is ≤ 40 % and Standard Deviation value (SD) is ≤ 18 %.A cell viability ≤ 40% corresponding to the cut-off value for positive control acceptance criteria was quantified for SDS 5% (26.58%). Also the standard deviation for positive control was inside the range of acceptability 0.86 ≤ 18%.
- Acceptance criteria met for variability between replicate measurements: test substance meets the acceptance criteria if the Standard Deviation value (SD) of the cell viability % (expressed as percentage compared to Negative Control) between the 3 tissue replicates is ≤ 18 %. (i.e. 4.14 % ≤ 18 %).

STATISTICAL ANALYSIS
No specific statistical analysis has been carried out except the standard deviation calculation.

Any other information on results incl. tables

O.D
SAMPLE	BIOLOGICAL REPLICATE 1			BIOLOGICAL REPLICATE 2			BIOLOGICAL REPLICATE 3
NC	0.8164	0.8624		0.8875		0.8759	0.8787	0.8879
SDS 5%	0.2226	0.2262		0.2266		0.2310	0.2419	0.2359
test item	0.8450	0.8818		0.9427		0.9192	0.9096	0.9270
O.D MEAN
SAMPLE	BIOLOGICAL REPLICATE 1			BIOLOGICAL REPLICATE 2			BIOLOGICAL REPLICATE 3
NC	0.8394			0.8817			0.8833
SDS 5%	0.2224			0.2288			0.2389
test item	0.8634			0.9310			0.9183
	VIABILITY						VIABILITY% MEAN±SD
	BIOLOGICAL REPLICATE 1		BIOLOGICAL REPLICATE 2		BIOLOGICAL REPLICATE 3
NC	96.69		101.57		101.75		100.00± 2.87
SDS 5%	25.85		26.36		27.52		26.58±0.86
test item	99.46		107.25		105.78		104.16±4.14

Applicant's summary and conclusion

Interpretation of results:

other: not skin irritant according to the CLP Regulation (EC n.1272/2008)

Conclusions:

Non-skin irritant.

Executive summary:

The present study has been conducted  in order to assess the skin irritation of the test item on the reconstructed human epidermis model EPISKIN-SMTMaccording to OECD TG 439 (July 2015).

Skin irritation was assessed at 15 min treatment followed by product washing and a post incubation period of 42h by using the MTT test method to quantify the residual cell viability.

Based on adopted prediction model and to MTT results (104.16 %) the test item was classified as not skin irritant according to UN GHS and according to the CLP Regulation (EC n.1272/2008).