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

Endpoint summary

Description of key information

In order to test the irritant/corrosive properties for skin and eyes of cC6O4 ammonium salt an in vitro skin irritation study and an in vitro eye irritation study (BCOP) were performed on cC6O4 ammonium salt, in compliance with test guidelines and GLP conditions.

In the human EPISKIN model, the relative absorbance value was slightly below the cut-off value, therefore the substance is considered a potential skin irritant.
Severe effects were observed in the BCOP assay.

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

Study period:

2009-03-19 until 2009-06-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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

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 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

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

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%

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

2009-03-26 to 2009-06-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline-conform study under GLP without deviations.

Qualifier:

according to guideline

Guideline:

other: The Bovine Corneal Opacity and Permeability Assay (BCOP) will be performed according to the INVITTOX (UK) protocol no. 98 "The Bovine Corneal Opacity and Permeability Assay", dated February 1994.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Draft OECD Guideline for Testing of Chemicals: The Bovine Corneal Opacity and Permeability (BCOP) Test Method for Identifying Ocular Corrosives and Severe Irritants (December, 2008).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz

Species:

other: in vitro test, using bovine cornea

Strain:

other: in vitro test, using bovine cornea

Details on test animals or tissues and environmental conditions:

not applicable, in vitro test

Vehicle:

physiological saline

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 100 mg at a volume of 1.0 mL on the surface of the corneas
- Concentration (if solution): 100 g/l


VEHICLE
- Amount(s) applied (volume or weight with unit): 1.0 ml per applicatiom
- Concentration (if solution): 0.9% (w/v)
- Lot/batch no. (if required): no data
- Purity: no data

Duration of treatment / exposure:

240 minutes (± 5 minutes)

Observation period (in vivo):

not applicable to in vitro testing

Number of animals or in vitro replicates:

Not applicable to in vitro testing; three bovine corneas used for each test item, negative and positive controls

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After incubation, test medium was rinsed off from the application side by changing cMEM buffer
- Time after start of exposure: 240 min


SCORING SYSTEM:

The following formula was used to determine the in vitro score of the negative control:
In vitro Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro score of the positive control and the test item:
In vitro Score = (opacity value – opacity valuenegative control + (15 x corrected OD490 value)
The in vitro score was calculated for each individual treatment and positive control cornea.
The mean in vitro score value of each treated group was calculated from the individual in vitro score values.

In vitro score Proposed in vitro irritation scale
0-3 non eye irritant
3.1-25 mild eye irritant
25.1-55 moderate eye irritant
55.1-80 severe eye irritant
>80.1 very severe eye irritant

TOOL USED TO ASSESS SCORE: This test is designed to measure the opacity of the cornea by quantifying the ability of light of pass through it. The permeability, as a result of the irritation potential of the test item, is determined using Na-fluorescein solution.

Irritation parameter:

in vitro irritation score

Value:

183.46

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: very severe irritant

Other effects / acceptance of results:

The mean "in vitro irritation score" was 183.46, thus much above the score of 80.1, which represents the minimum score for the data interpretation "very severe irritating".

Table 1: Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity *	Permeability at 490 nm (OD490)*	In vitro Score	Mean in vitro Score	Proposed in vitro Irritation Scale
Negative control (mean of 3 tests)	0	0.143		2.15	No eye irritant
Positive Control	104	-0.063	103.05
Positive Control	113	-0.034	112.49	105.73	Very severe eye irritant
Positive Control	101	0.044	101.66
cC6O4	214	1.985	243.77
cC6O4	130	2.232	163.48	183.46	Very severe eye irritant
cC6O4	109	2.276	143.14

*corrected values

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item cC6O4 ammonium salt is considered to be a very severe eye irritant. As the observed effects caused by the test item exceeded by far the effects caused by the positive control substance, a worst case classification is suggested, i.e. CLP classification H318, eye damage 1.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of cC6O4 ammonium salt by means of the BCOP assay using fresh bovine corneas.

After a first opacity measurement of the fresh bovine corneae (t0), about 100 mg of the test item cC6O4 ammonium salt, the positive, and the negative controls were applied to corneas and incubated for 240 minutes at 32 ± 2 °C in complete medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneas and opacity was measured again (t240). After the opacity measurements permeability of the corneas was determined while application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 2 °C in a horizontal position. The coming out liquid was measured spectrophotometrically.

With the negative control (0.9% NaCl solution) neither an increase of opacity nor permeability of the corneas could be observed. The in vitro score was calculated as 2.15. The positive control (10% (v/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneas and therefore, is classified as very severe eye irritant. The in vitro score was calculated as 105.73. The test item cC6O4 ammonium salt caused opacity and permeability of the corneas compared with the results of the negative control. The calculated in vitro score was 183.46 and therefore, the test item was classified as very servere eye irritant.

As the observed effects caused by the test item exceeded by far the effects caused by the positive control substance, a worst case classification is suggested, i.e. CLP classification H318, eye damage 1.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In order to test the irritant/corrosive properties for skin and eyes of cC6O4 ammonium salt an in vitro skin irritation study and an in vitro eye irritation study (BCOP) were performed on cC6O4 ammonium salt. The studies were guidelines conform and performed under GLP conditions.

 

In the skin irritation study three tissues of the human skin model EPISKIN were treated with either the test item cC6O4 ammonium salt, the negative or the positive control for 15 minutes. After treatment with cC6O4 ammonium salt the relative absorbance values were decreased to 49.2%. This value is slightly below the threshold for irritancy of ≤ 50% and therefore the test item was considered to possess an irritant potential.

In the eye irritation study, performed to assess the corneal irritation and damage potential of cC6O4 ammonium salt using fresh bovine corneas, the test item caused opacity and permeability of the corneas compared with the results of the negative control and therefore cC6O4 ammonium salt was considered to be a very severe eye irritant.

Effects on skin irritation/corrosion: irritating
Effects on eye irritation: highly irritating

Justification for classification or non-classification

Basing on the results above reported, REGULATION (EC) No 1272/2008 ( EU Regulation on Classification, Labelling and Packaging of substances and mixtures) would indicates the following for cC6O4 ammonium salt:

 

Classification

Skin corrosion/irritation: Skin Irritation Category 2

Hazard statement: H315: Causes skin irritation

 

Serious eye damage/ eye irritation: Eye damage 1

Hazard statement: H318: Causes serious eye damage.