Acetic acid, 2,2-difluoro-2-[[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy]-, ammonium salt (1:1)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-03-19 until 2009-06-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPlSKlN and EpiDerm assays and on the Skin Integrity Function Test (Altern Lab Anim. 2007 Dec; 35 (6): 559-601).

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: European Commission: Institute for Health an Consumer Protection and European Centre for the Validation of Alternative Methods (ECVAM): Statement on the validity of in-vitm tests for skin irritation (2007).

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Human Skin Model Test, December 2007.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz

Test material

Test material information

Constituent 1

Reference substance name:

cC6O4 ammonium salt

IUPAC Name:

cC6O4 ammonium salt

Constituent 2

Reference substance name:

Acetic acid, 2,2-difluoro-2-[[2,2,4,5- tetrafluoro-5-(trifluoromethoxy)-1,3- dioxolan-4-yl]oxy]-, ammonium salt (1:1)

IUPAC Name:

Acetic acid, 2,2-difluoro-2-[[2,2,4,5- tetrafluoro-5-(trifluoromethoxy)-1,3- dioxolan-4-yl]oxy]-, ammonium salt (1:1)

Constituent 3

Reference substance name:

ammonium 2,2-difluoro-2-{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate

EC Number:

682-238-0

Cas Number:

1190931-27-1

Molecular formula:

C6H4F9NO6

IUPAC Name:

ammonium 2,2-difluoro-2-{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetate

Details on test material:

- Name of test material (as cited in study report): cC6O4
- Substance type: pure substance
- Physical state: solid
- Analytical purity: 96.2%
- Impurities (identity and concentrations): NH4F=0.021% (w/w), NH4Cl=0.31% (w/w), H2O=0.328% (w/w), the organic impurities are dioxolan based and represent the 0.8% by mol on total mol of organic phase of sample
- Composition of test material, percentage of components: not reported
- Isomers composition: not reported
- Purity test date: not reported
- Lot/batch No.: 150/31
- Expiration date of the lot/batch: 31 December 2020
- Stability under test conditions: Stable in water and saline at room temperature for days
- Storage condition of test material: at room temperature

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN kits

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

water

Remarks:

deionised water

Details on test system:

CELL CULTURE FOR IN VITRO TEST:
EPISKIN kits are purchased from Skinethic Laboratories (06000 Nice, France).
The EPISKIN tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EPISKIN tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EPISKIN tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Harlan CCR on April 08, 2009. On day of receipt EPISKIN tissues were transferred to 12-well plates with maintenance medium.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 15 mg of the test item were applied to each tissue and wetted with 15 μL deionised water
- Concentration (if solution): 1000 g/l

VEHICLE
- Amount(s) applied (volume or weight with unit): 15 μL deionised water

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C, 5 ± 0.5% CO2
- Temperature of post-treatment incubation (if applicable):


REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps:
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength:
- Filter:
- Filter bandwidth:

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: triplicate tissues

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. if the mean TER value is less than or equal to 5 kΩ and the skin disk is obviously damaged, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, but the mean disc dye content is greater than or equal to the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. if the mean TER value obtained for the test substance is greater than 5 kΩ, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, and the mean disc dye content is well below the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430:

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Test system

Duration of treatment / exposure:

15 +/- 1 min direct exposure of the cell culture to the test item (after this 42 hours incubation)

Observation period:

not applicable, direct readings of the assay results

Number of animals:

No animals used. Three tissues were used, from each two replicate wells were tested per test substance concentration and controls.

Details on study design:

not applicable

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: Relative absorbance (% of negative control)

Value:

49.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

(3% of control)

Remarks on result:

other:

Remarks:

Basis: mean tissue 1, 2, and 3. Time point: 15 min exposure. Max. score: 62.83. Reversibility: not assessed.

Other effects / acceptance of results:

The relative absorbance in % of negative control is used for quantifying the potential skin irritating effect of the tested substances. After treatment with the test item cC6O4 the relative absorbance values were decreased to 49.2%. This value is slightly below the threshold for irritancy of ≤ 50%

Any other information on results incl. tables

Table 1: Results after treatment with cC604

Dose group	Treatment Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Rel. Absorbance [% of Negative Control]**
Negative Control	15 min	0.8993	0.8653	0.7961	0.8536	100
Positive Control	15 min	0.0371	0.0247	0.0156	0.0258	3.0
cC6O4	15 min	0.3922	0.3656	0.5016	0.4198	49.2

* Mean of two replicate wells after blank correction

** relative absorbance [rounded values]: (absorbance ) 100 x (absorbance test item) / (absorbance negative control)

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

As a conclusion for this in-vitro study it can be stated that under the experimental conditions reported, the test item cC6O4 ammonium salt is irritant to skin. The in-vivo prediction would be an EU classification as Xi; R38, and a CLP classification as H315, Skin. irrit. 2.

Executive summary:

This in vitro study was performed to assess the irritation potential of cC6O4 ammonium salt by means of the Human Skin Model Test.

Three tissues of the human skin model EPISKIN were treated with either the test item, the negative or the positive control for 15 minutes. 15 mg of the test item were applied to each tissue and wetted with 15 μL deionised water. 15 μL of either the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue. After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 0.6 for the 15 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.0% thus ensuring the validity of the test system. After treatment with the test item cC6O4 ammonium salt the relative absorbance values were decreased to 49.2%. This value is slightly below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Note: For the current test, an irritation potential of a test item according to EU CLP classification, skin irrit. 2, H315 is predicted if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.