Tetradecafluorohexane

Endpoint summary

Description of key information

Data were generated using standardized methods under GLP for skin irritation and corrosion, and for eye irritation. No adverse effect were observed during these assays.

As tetradecafluorohexane is not corrosive nor irritant for skin and not irritant for eyes, no data was generated for eye corrosion.

Indication of absence of irritation on airways is provided by inhalation assays described in acute toxicity section (7.2.2.).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

Test system
EpiDerm Skin Model (EPI-200, Lot no.: 28356, kit A, Appendix 4).
The model 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 EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
Rationale
Recommended test system in international guidelines (OECD and EC). Source
MatTek Corporation, Ashland MA, U.S.A.

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

3 minutes & 1 hour

Number of replicates:

2 x 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 minutes

Value:

ca. 97

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

11

Remarks on result:

other: No indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

5.6

Remarks on result:

other: No indication of corrosion

Other effects / acceptance of results:

Tétradécafluorohexane was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit <2.8) and the laboratory historical control data range (See Appendix 3). The mean relative tissue viability following the 1-hour exposure to the positive control was 5.6%.
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was
< 17%, indicating that the test system functioned properly (Appendix 1, Table 3).

Table 1

Mean Absorption in the in vitro Skin Corrosion Test with Tétradécafluorohexane

 

	A(OD570)	3-minute application B(OD570)         Mean (OD570)			SD	1-hour application A (OD570)  B(OD570)                                    Mean (OD570)				SD
Negative control	1.609	1.929	1.769	±	0.227	1.838	1.843	1.841	±	0.003
Tétradécafluorohexane	1.774	1.656	1.715	±	0.084	1.831	1.650	1.741	±	0.129
Positive control	0.247	0.131	0.189	±	0.082	0.089	0.115	0.102	±	0.018

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0423). Isopropanol was used to measure the background absorption.

Table 2

Mean Tissue Viability in the in vitro Skin Corrosion Test with Tétradécafluorohexane

 

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Tétradécafluorohexane	97	95
Positive control	11	5.6

 

Table 3

Coefficient of Variation between Tissue Replicates

 

	3 minute	1 hour
Negative control	17	0.3
Tétradécafluorohexane	6.7	9.9
Positive control	47	22

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Individual OD Measurements at 570 nm

	3-minute application(OD570) A              B		1-hour application(OD570) A              B
Negative control OD570 measurement 1	1.6578	1.9712	1.8766	1.9152
OD570 measurement 2	1.6490	1.9674	1.8973	1.8840
OD570 measurement 3	1.6464	1.9758	1.8675	1.8570
Test itemOD570 measurement1	1.8177	1.6722	1.9150	1.7002
OD570 measurement 2	1.8213	1.6912	1.8495	1.6773
OD570 measurement 3	1.8107	1.7306	1.8568	1.6985
Positive control OD570 measurement 1	0.2894	0.1751	0.1334	0.1601
OD570 measurement 2	0.2863	0.1716	0.1327	0.1559
OD570 measurement 3	0.2915	0.1722	0.1288	0.1567

OD = Optical density

Duplicate exposures are indicated by A and B.

Historical Control Data for in vitro Skin Corrosion Studies

	Negative control		Positive control
	3-minutetreatment (OD570)	1-hourtreatment (OD570)	3-minutetreatment (OD570)	1-hourtreatment (OD570)
Range	1.258 – 2.615	1.371 – 2.371	0.0172 – 0.56	0.046 – 0.339
Mean	1.80	1.82	0.19	0.14
SD	0.26	0.22	0.09	0.05
n	111	110	106	103

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2014 to November 2017.

Interpretation of results:

GHS criteria not met

Conclusions:

Tétradécafluorohexane is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.

Executive summary:

The objective of this study was to evaluate Tétradécafluorohexane for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of Tétradécafluorohexane was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 57 A of Tétradécafluorohexane was a clear colourless liquid. Tétradécafluorohexane was applied undiluted (50 µL) directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 5.6% after the 1-hour exposure.The absolute mean OD570(optical density at 570 nm) of the negative control tissues waswithin the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upperacceptance limit£2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was£17%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Tétradécafluorohexane compared to the negative control tissues was 97% and 95%, respectively. Because the mean relative tissue viability for Tétradécafluorohexane was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment Tétradécafluorohexane is considered to be not corrosive.

In conclusion, Tétradécafluorohexane is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

may - august 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 µL

Duration of treatment / exposure:

15 +/- 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Controls:

yes

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 108

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Table 1. Mean Absorption in theIn VitroSkin Irritation Test with Tétradécafluorohexane

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	0.960	0.937	1.015	0.971	±	0.040
Tétradécafluorohexane	1.146	0.968	1.044	1.053	±	0.089
Positive control	0.073	0.078	0.061	0.070	±	0.009

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

 

Table2           
Mean Tissue Viability in theIn VitroSkin Irritation Test withTétradécafluorohexane

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	4.2
Tétradécafluorohexane	108	9.2
Positive control	7.3	0.9

Individual OD Measurements at 570 nm

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	1.0123 0.9921	0.9900 0.9680	1.0793 1.0356
Tétradécafluorohexane OD570measurement 1 OD570measurement 2	1.1670 1.2083	0.9851 1.0345	1.1226 1.0504
Positive control OD570measurement 1 OD570measurement 2	0.1118 0.1180	0.1221 0.1175	0.0982 0.1070

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Interpretation of results:

GHS criteria not met

Conclusions:

Tétradécafluorohexane is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

The objective of this study was to evaluate Tétradécafluorohexane for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM^TM). The possible skin irritation potential of Tétradécafluorohexane was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 57 A of Tétradécafluorohexane was a clear colourless liquid. Tétradécafluorohexane was applied undiluted (25 µL) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Tétradécafluorohexane compared to the negative control tissues was 108%. Since the mean relative tissue viability for Tétradécafluorohexane was above 50% after 15 ± 0.5 minutes treatment Tétradécafluorohexane is considered to be non-irritant.

The positive control had a mean cell viability of 7.3% after 15 ± 0.5 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 10%, indicating that the test system functioned properly.

In conclusion, Tétradécafluorohexane is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report andshould not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted October 09, 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item information
Identification: Tétradécafluorohexane
Appearance: Clear colourless liquid
Batch: 57 A
Purity/Composition: 99.25%
Test item storage: At room temperature protected from light
Stable under storage conditions until: 21 February 2020 (expiry date)
Additional information
Test Facility test item number: 209384/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Stability at higher temperatures: Stable

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands),
where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 %µL

Duration of treatment / exposure:

10 +/- 1 minutes

Duration of post- treatment incubation (in vitro):

120 +/- 10 minutes

Number of animals or in vitro replicates:

3

Irritation parameter:

in vitro irritation score

Value:

ca. -0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

1.6

Positive controls validity:

valid

Remarks:

40

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Value:

ca. -0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

1.8

Positive controls validity:

valid

Remarks:

16

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Value:

ca. 0.008

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

-0.0016

Positive controls validity:

valid

Remarks:

1.625

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. Altough the negative control treated corneas were translucent, all results are within the acceptability range therefore this has no impact on the study result. The mean in vitro irritancy score of the positive control (Ethanol) was 40 and within two standard deviations of the current historical positive control mean (Appendix 3, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Tétradécafluorohexane was tested neat.

Table 1of Appendix 1summarizes the opacity, permeability and in vitro irritancy scores of Tétradécafluorohexane and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Table 2- 5.

The individual in vitro irritancy scores for the negative controls ranged from 0.8 to 2.7. The corneas treated with the negative control item were translucent after the 10 minutes of treatment. All values were within the historical control database. The individual positive control in vitro irritancy scores ranged from 31 to 48 (Appendix 2,Table 5). The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with Tétradécafluorohexane showed opacity values ranging from -0.9 to

-0.5 and permeability values ranging from 0.006 to 0.011. The corneas were translucent after the 10 minutes of treatment with Tétradécafluorohexane. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.7 to -0.4 after 10 minutes of treatment with Tétradécafluorohexane.

Table 1

Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity1	Mean Permeability1	Mean In vitro Irritation Score1, 2
Negative control	1.8	-0.016	1.6
Positive control (Ethanol)	16	1.625	40
Test item	-0.7	0.008	-0.5

1        Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

2        In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Individual Opacity, Permeability and in vitro Score

Table 2 Opacity Score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity1		Negative control corrected Final Opacity2	Mean Final Opacity
Negative control	4.7	6.2	1.4			1.8
	4.5	5.5	1.0
	3.9	6.8	2.9
Positive control	2.0	18.3	16.3	14		16
	3.5	20.8	17.4	16
	4.4	23.3	18.9	17
Test item	3.7	4.8	1.2	-0.6		-0.7
	3.9	5.2	1.3	-0.5
	4.4	5.3	0.9	-0.9

Calculations are made without rounding off.

1Final Opacity = Opacity after treatment – Opacity before treatment.

2Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control.

Table 3

Permeability Score Individual Values (Uncorrected)

 

Treatment	Dilutionfactor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative control
Negative control	1	-0.017	-0.017	-0.017	-0.017	-0.017	-0.016
	1	-0.016	-0.016	-0.016	-0.016	-0.016
	1	-0.016	-0.016	-0.016	-0.016	-0.016
Positive control	6	0.272	0.276	0.277	0.275	1.650
	6	0.349	0.345	0.342	0.345	2.072
	1	0.931	0.943	0.945	0.940	0.940
Test item	1	-0.010	-0.010	-0.011	-0.010	-0.010
	1	-0.009	-0.010	-0.011	-0.010	-0.010
	1	-0.010	-0.008	0,001	-0.009	-0.009

Calculations are made without rounding off.

Table 4

Permeability Score Individual Values (Corrected)

 

Treatment	Dilution factor	Negative control correctedOD4901^1	Negative control correctedOD4902^1	Negative control correctedOD4903^1	Negative control correctedOD490 Average	Negative control corrected final OD490	Average OD
Positive control	6	0.288	0.292	0.293	0.291	1.748	1.625
	6	0.365	0.361	0.358	0.362	2.170
	1	0.947	0.959	0.961	0.956	0.956
Test item	1	0.006	0.006	0.005	0.006	0.006	0.008
	1	0.007	0.006	0.005	0.006	0.006
	1	0.006	0.008	0.017	0.011	0.011

Calculations are made without rounding off.

1OD490values corrected for the mean final negative control permeability (-0.016).

 

Table 5

In Vitro Irritancy Score

 

Treatment	Final Opacity2	Final OD  2 490	In vitro Irritancy Score1
Negative control	1.4	-0.017	1.2
	1.0	-0.016	0.8
	2.9	-0.016	2.7
Positive control	14	1.748	41
	16	2.170	48
	17	0.956	31
Test item	-0.6	0.006	-0.5
	-0.5	0.006	-0.4
	-0.9	0.011	-0.7

1    In vitro irritancy score (IVIS) = opacity value + (15 x OD490value).

2    Positive control and test item are corrected for the negativecontrol.

Table 6

Historical Control Data for the BCOP Studies

 

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-2.9 – 3.0	-0.034 – 0.100	-2.8 – 3.0	28.0 – 110.9
Mean	0.25	0.00	0.31	55.03
SD	1.13	0.01	1.19	15.08
n	118	118	118	94

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2015 to May 2018.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since Tétradécafluorohexane induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Tétradécafluorohexane as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of Tétradécafluorohexane was tested through topical application for 10 minutes.

The study procedures described in this report were based on the most recent OECD guideline. Batch 57 A of Tétradécafluorohexane was a clear colorless liquid with a purity of 99.25%.

The test item was applied as it is (750 µL) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limitsof the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 40 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Tétradécafluorohexane did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.5 after 10 minutes of treatment.

In conclusion, since Tétradécafluorohexane induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

No irritation nor corrosion potential was detected in in vitro assays, nor in in vivo studies carried on for other purposes.