Ammonium undecafluorohexanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted in 2016

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium undecafluorohexanoate

EC Number:

244-479-6

EC Name:

Ammonium undecafluorohexanoate

Cas Number:

21615-47-4

Molecular formula:

C6H4F11NO2

IUPAC Name:

2,2,3,3,4,4,5,5,6,6,6-Undecafluorohexanoic acid, ammonium salt (1:1)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (EPI-200)

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™(EPI-200) (MatTek Corporation, Ashland, MA, USA)
- Tissue batch number: 28302
- Production date: 22 Mar 2018


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 min exposure); 37 °C (60 ± 1 min exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were rinsed with PBS 20 times in order to remove any residual test material.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mL/well
- Incubation time: 180 ± 5 min
- Spectrophotometer: microplate reader (FLUOstar OPTIMA, BMG LABTECH)
- Wavelength: 570 nm


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.708 ± 0.063 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.42 h (acceptance criteria: 3.68-8.02 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance showed no reducing capacity 1 h after MTT incubation, no additional test with freezekilled tissues had to be performed.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if the viability after 3 min exposure is greater than 50% and the viability after 1 h exposure is greater than 15%.
- The test substance is considered to be corrosive to skin if the viability after 3 min exposure is less than 50%, or if the viability after 3 min exposure is greater than or equal to 50% and the viability after 1 h exposure is less than 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg moistened with 25 µL distilled water

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration : 8 N

Duration of treatment / exposure:

3 min and 60 ± 1 min

Number of replicates:

duplicates for each treatment and control group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

3 min exposure

Value:

94.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 min exposure

Value:

5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The test substance showed no reducing capacity 1 hour after MTT incubation.
- Colour interference with MTT: The test substance did not change colour, when mixed with deionised water and thus passed the colour interference pre-test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (1.941 and 2.047).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 hour, was < 15% (1.4%) compared to the negative control.
- Acceptance criteria met for variability between replicate measurements: The Coefficient of Variation (CV) in the range 20 - 100% viability between tissue replicates was ≤ 30% (values between 0.1% and 11.74%)

Any other information on results incl. tables

Table 1. Results of skin corrosion test

		3-min exposure							60-min exposure
		OD a)			Cell viability (%) b)		SD c)	CV	OD a)			Cell viability (%) b)		SD c)	CV
Group	Tissue no.		Mean		Mean					Mean		Mean
Negative control (Distilled water)	1	1.937	1.939	1.941	99.9	100	0.14	0.1	2.026	2.03	2.047	99.2	100	1.13	1.1
		1.937							2.031
		1.942							2.032
	2	1.938	1.942		100.1				2.07	2.063		100.8
		1.945							2.063
		1.942							2.056
Positive control (KOH 8N)	1	0.113	0.112	0.111	5.8	5.7	0.14	2.5	0.029	0.029	0.028	1.4	1.4	0.07	5.1
		0.111							0.029
		0.113							0.03
	2	0.108	0.109		5.6				0.025	0.026		1.3
		0.111							0.025
		0.109							0.027
Test substance (APFHx)	1	1.68	1.671	1.833	86.1	94.4	11.74	12.4	0.103	0.103	0.102	5	5	0.07	1.4
		1.669							0.104
		1.665							0.103
	2	1.983	1.994		102.7				0.095	0.1		4.9
		1.997							0.109
		2.002							0.096

OD: optical density; SD: standard deviation; CV: coefficient of variation

a) OD value corrected by mean blank OD

b) Cell viability of negativ control was regarded as 100%.

c) The SD was calculated from the cell viabilities of each tissue insert (n=2).

Table 2. Historical control data (Jan 2016 - Feb 2018)

	OD
	3- min exposure		60-min exposure
	Negative control	Positive control	Negative control	Positive control
Mean	1.9	0.2006	1.852	0.065
SD	0.192	0.069	0.191	0.031
Max	2.331	0.371	2.176	0.113
Min	1.635	0.028	1.573	0.024
Number of tests	20		20

OD: optical density

SD: standard deviation

Applicant's summary and conclusion

Interpretation of results:

other: Skin Corr. 1B/C according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the reconstructed human epidermis test cell viability after 3-min and 60-min exposure to the test substance were 94.4% and 5.0%, respectively.