Ammonium undecafluorohexanoate

Endpoint summary

Description of key information

Skin irritation/corrosion (OECD 431): corrosive

Eye irritation /corrosion (OECD 405): corrosive

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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 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

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

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%)

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Feb - 05 Apr 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted in 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

adopted in 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

adopted in Nov 2000

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted in 2015

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Remarks:

(SPF-Quality)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: 2.049 - 2.330 kg (range)
- Housing: individually in cages with perforated floors (56x44x37.5 cm)
- Diet: standard laboratory rabbit diet (Charles River Breeding and Maintenance Diet for Rabbits, Altromin, Lage, Germany) approx. 100 g per day, additionally pressed hay three times a week
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.5 - 24.7
- Humidity (%): 29 - 78
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: The other eye of each animal remained untreated and served as the reference control.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL

Duration of treatment / exposure:

single application without washing

Observation period (in vivo):

28 days
reading time points: 1, 24, 48 and 72 h and 7, 14, 21 days (and/or 28 days in the sentinel animal)

Number of animals or in vitro replicates:

3 males

Details on study design:

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: fluorescein; Where standard lighting was considered inadequate for observing minor effects, eye examinations were performed using an ophthalmic examination lamp.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: a 20% aqueous solution of the test substance was used

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: a 20% aqueous solution of the test substance was used

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Remarks on result:

other: a 20% aqueous solution of the test substance was used

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: a 20% aqueous solution of the test substance was used

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 72 h

Remarks on result:

other: a 20% aqueous solution of the test substance was used

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: a 20% aqueous solution of the test substance was used

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.67

Max. score:

3

Reversibility:

fully reversible within: 28 days

Remarks on result:

other: a 20% aqueous solution of the test substance was used

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 21 days

Remarks on result:

other: a 20% aqueous solution of the test substance was used

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

fully reversible within: 21 days

Remarks on result:

other: a 20% aqueous solution of the test substance was used

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: a 20% aqueous solution of the test substance was used

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: a 20% aqueous solution of the test substance was used

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: a 20% aqueous solution of the test substance was used

Irritant / corrosive response data:

The corneal injury consisted of opacity (maximum grade 2) in all animals and epithelial damage (maximum fluor area 35%, 65% and 50% of the corneal area). In 2/3 animals corneal injury was reversible within 7 days and persisted until day 21 in the remaining animal. lridial irritation (grade 1) was observed in all animals and was reversible within 72 hours in 1/3 animals and within 7 days in the two remaining animals. The irritation of the conjunctivae consisted of redness (maximum grade 3), chemosis (maximum grade 4) and discharge (maximum grade 3). Redness had completely resolved within 21 and 28 days in 2/3 animals, respectively, and persisted until termination (day 21) in the remaining animal. Chemosis and discharge had completely resolved within 7 days in all animals.

Other effects:

- Lesions and clinical observations: As a result of the corneal injury, pannus (neovascularisation of the cornea) was apparent in 1/3 animals 7 and 21 days after instillation. Reduced elasticity of the eyelids was noted in all animals 24 and/or 48 hours and/or 7 days after instillation.
- Other observations: No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Table 1: Results of eye irritation study.

Alteration	Animal No.	Time (h)
		1	24	48	72	mean 24/48/72 h	reversible within
Cornea (opacity)	1	1	2	1	1	1.3	7 days
	2	1	1	1	1	1.0	7 days
	3	1	1	1	1	1.0	Not reversible within 21 days
Area of corneal opacity *	1	1	1	2	1	-	-
	2	3	3	3	2	-	-
	3	3	2	2	2	-	-
Iris	1	0	0	1	1	0.7	7 days
	2	1	1	1	0	0.7	72 h
	3	1	1	1	1	1.0	7 days
Conjunctivae Redness	1	2	2	3	3	2.7	28 days
	2	2	3	3	3	3.0	Not reversible within 28 days
	3	2	3	3	3	3.0	21 days
Conjunctivae Chemosis	1	4	3	2	1	2.0	7 days
	2	3	3	2	1	2.0	7 days
	3	3	2	2	1	1.7	7 days

*Area of cornea involved:

0 = No ulceration or opacity

1 = One quarter or less but not zero

2 = Greater than one quarter, but less than half

3 = Greater than half, but less than three quarters

4 = Greater than three quarters, up to whole area

 

 

 

 

 

 

 

 

 

 

 

 

Interpretation of results:

other: Eye damage Cat. 1 according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of this eye irritation study in rabbits the test substance was corrosive to the eye. Although a 20% aqueous solution of the test substance was used in this study, results are considered valid as corrosive properties can also be assumed for the pure test substance. Thus, the test substance is classified as Eye Dam. 1 (H318) according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

In vitro

The skin corrosion potential of the test substance was assessed by an in vitro skin corrosion test using a human skin model (Reconstructed human Epidermis test method) according to OECD guideline 431 and in compliance with GLP (Miyaura, 2018). Each of two human skin tissues (EpiDerm™) were treated with 25 mg of the test substance moistened with 25 µL distilled water, the negative control (distilled water) or the positive control (potassium hydroxide 8 N) for 3 and 60 min, respectively. The cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The relative mean tissue viabilities obtained after 3 and 60 min treatment with the test substance compared with the negative control tissues were 94.4% and 5.0%, respectively. As the mean relative tissue viability for the test substance was below 15% after 60 min treatment the test substance was considered to be corrosive to the skin. The optical pre-experiment (colour interference pre-experiment) did not lead to a change in colour by the test substance. Additionally, optical evaluation of the MTT-reducing capacity of the test substance after 1 hour incubation with MTT-reagent did not show blue colour. The positive control, potassium hydroxide, had a mean cell viability of 1.4% (required ≤ 15%) after 60 min exposure; showing the validity of the test system. All other acceptability criteria were met. Based on the results of this study, the test substance was corrosive to the skin under the conditions of the test. This leads to the classification Skin Corrosion 1B/C.

In vivo

The skin irritation potential of a 20% solution of the test substance was determined in an in vivo skin irritation/corrosion test performed in three albino rabbits according to OECD guideline 404 and in compliance with GLP (Teunissen, 2004). In a first step, one rabbit was exposed to three samples of 0.5 mL of the test substance on the clipped skin using semi-occlusive dressings. After exposure periods of 3 min, 1 h and 4 h, respectively, the patch was removed and the treated areas were rinsed with water. Local skin irritation effects were scored 1, 24, 48 and 72 h and 7 days after patch removal using the Draize scoring system. Based on the absence of severe skin reactions, two further animals were exposed for 4 hours to the test substance in a next step. After 4 h exposure, well-defined erythema (score 2) was observed in 3/3 animals, fully reversible in all animals within 7 days. Very slight edema (score 1) was observed in 3/3 animals, reversible in all animals within 48 h. Mean scores for 24, 48 and 72 h were 1.7, 1.7 and 1.7 for erythema and 0.3, 0.3 and 0.3 edema for the 3 rabbits, respectively. Since the study was conducted with a 20% aqueous solution of the test substance, this study cannot be used for classification purposes.

 

Eye, in vivo

The eye irritation/corrosion potential of a 20% solution of the test substance was assessed in three albino rabbits according to OECD guideline 405 and in compliance with GLP (Teunissen, 2003). 0.1 mL of the test substance was instilled in one eye of each animal. The other eye remained untreated and served as control. The eyes were examined and scored according to Draize 1, 24, 48 and 72 h and 7, 14, 21 and/or 28 days after application. After instillation of the test substance corneal opacity (maximum grade 2) and epithelial damage (maximum fluor area 35%, 65% and 50% of the corneal area) was observed in all animals. As a result of the corneal injury, pannus (neovascularisation of the cornea) was apparent in one animal up to 21 days after instillation. In 2/3 animals corneal injury was reversible within 7 days. lridial irritation (grade 1) was observed in all animals and was reversible within 72 h in 1/3 animals and within 7 days in the two remaining animals. The irritation of the conjunctivae consisted of redness (maximum grade 3), chemosis (maximum grade 4) and discharge (maximum grade 3). Redness had completely resolved within 21 and 28 days in 2/3 animals, respectively, and persisted until termination (Day 21) in the remaining animal. Chemosis and discharge had completely resolved within 7 days in all animals. Reduced elasticity of the eyelids was noted 24 and/or 48 hours and/or 7 days after instillation. Mean scores for 24, 48 and 72 h were 1.33, 1 and 1 for corneal opacity, 0.67, 0.67 and 1 for iris score, 2.67, 3 and 3 for conjunctivae score and 2, 2 and 1.67 for chemosis score for the 3 animals, respectively. Based on the results of this study, the test substance is considered to be corrosive to the eye under the conditions of the test. Although a 20% aqueous solution of the test substance was used in this study, corrosive properties can also be assumed for the pure test substance. This leads to the classification Eye Dam. 1.

Justification for classification or non-classification

The available data on skin and eye irritation/corrosion of the test substance meet the criteria for classification as Skin Corr. 1B/C (H314) and Eye Dam. 1 (H318) according to Regulation (EC) No 1272/2008.