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Description of key information

The test substance is corrosive: in contact with water it releases F- ions, known to cause deep tissue damage and pain.  Both an in vitro skin corrosion test and local skin damage in a mouse sensitisation study indicated corrosive action leading to classification, and an in vitro test for eye irritation found severe irritant activity.

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:
20 March 2008-6 June 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline no. 431: In Vitro Skin Corrosion: Human Skin Model Test (adopted 13 April 2004).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
not specified
Details on test animals and environmental conditions:
Test system
EpiDerm Skin Model (EPI-200, Lot no.: 10526 kit H).
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).

Tissues
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

DMEM (Dulbecco’s Modified Eagle’s Medium)
Supplemented DMEM medium, serum-free.

MTT medium
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM).

Environmental conditions
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 71 - 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.0 - 36.7°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity (with a maximum of 9%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Type of coverage:
open
Preparation of test site:
other: not applicable
Vehicle:
other: moistened with Milli-Q water
Controls:
other: negative control: Milli-Q water
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg, moistened with 25 µl Milli-Q water
The test substance was spread to match the size of the tissue.
Duration of treatment / exposure:
3 minutes and 1 hr
Observation period:
None
Number of animals:
4 tissues per test substance
Details on study design:
The skin tissues were kept in the refrigerator the day they were received. The next day, before the assay is started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The plates were incubated for 1.75 hour at 37.0 ± 1.0°C. The level of the DMEM medium is just beneath the tissue. The medium was replaced with fresh DMEM medium just before LiPF6 was applied. The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a three minutes exposure to LiPF6 at room temperature and two for a one hour exposure at 37.0 ± 1.0°C. LiPF6 (25 mg) with 25 µl Milli-Q water was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 µl Milli-Q water (negative control) and with 50 µl 8N KOH (positive control), respectively. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 µl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.
Cell viability measurement: the DMEM medium was replaced by 300 µl MTT-medium and tissues were incubated for 3 h at 37°C in air containing 5 ± 0.5% carbon dioxide. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 540 nm in triplicate with the Multiskan Spectrum (Thermo Labsystems).

Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

Irritation / corrosion parameter:
other:
Value:
103
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 min. Reversibility: no data. Remarks: score is percentage of control. (migrated information)
Irritation / corrosion parameter:
other:
Value:
6
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 1 hr. Reversibility: no data. Remarks: score is percentage of control. (migrated information)
Irritant / corrosive response data:
Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3 minutes and 1 hour treatment with LiPF6 compared to the negative control tissues was 103% and 6% respectively. Since the mean relative tissue viability for LiPF6 was below 15% after 1 hour treatment it is considered to be corrosive.

The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability of the 3 minutes exposure of the positive control was 5%. The maximum inter tissue variability in viability between two tissues treated identically was less than 21% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 12%. It was therefore concluded that the test system functioned properly.

Other effects:
None.
Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
It is concluded that this test is valid and that LiPF6 is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

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:
12 March 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The test has been performed based on international recommendations. Although the HET-CAM test method is not yet validated, the EU national regulatory authorities accept positive outcomes (severe eye irritants) of this test method for classification and labelling severe eye irritants. Where a negative result is obtained, and in vivo test is subsequently required.
Qualifier:
no guideline available
Principles of method if other than guideline:
The study procedures described in this report were based on the following documents and follows international recommendations (ref. 1-5)

- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) test method, March 2006.
- INVITTOX protocol 47. The HET-CAM test - Method of Spielmann and Liebsch, January 1992.

ref 1: Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 405, "Acute Eye Irritation / Corrosion", Paris Cedex, 2002.
ref 2: UN 2003. Globally Harmonized System of Classification and Labelling of Chemicals (GHS). United Nations Publications: New York & Geneva.
ref 3: EC 2004. Commission directive 2004/73/EC adapting to technical progress for the 29th time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions related to classification, packaging and labelling of dangerous substances. Official Journal of the European Union L152:1-316. April 2004.
ref 4: EC 2004. Manual of Decisions for Implementation of the 6th and 7th amendments to Directive 67/548 on Dangerous Substances (Directives 79/831/EEC and 92/32/EEC). July 2004.
ref 5: EPA 1998. Health Effects Test Guideline, OPPTS 870.2400 Acute Eye Irritation. EPA 712-C-98-195. Washington, DC: U.S. Environmental Protection Agency.

The HET-CAM assay measures three important components which are predictive for conjunctival injury of the eye, heamorrhage, vessel lysis and coagulation.



GLP compliance:
yes (incl. certificate)
Species:
other: Fresh, fertilised brown leghorn chicken eggs
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eggs were obtained from a non-GLP chicken-breeding center (Het Anker Ochten, the Netherlands).
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
Liquids
Three eggs per treatment group were treated for 300 seconds with 0.3 ml of the negative or positive control solutions. The appearance of haemorrhage, vessel lysis and coagulation on the CAM was monitored and recorded over this 300-second period.

Solids
Three eggs were treated for 20 seconds with 300 mg LiPF6. After the treatment period the eggs were carefully rinsed with approximately 5 ml of tepid water (Milli-RO water). The appearance of haemorrhage, vessel lysis and coagulation on the CAM was monitored and recorded over the following 280-second period.
Duration of treatment / exposure:
20 seconds
Observation period (in vivo):
280 seconds
Number of animals or in vitro replicates:
3 eggs per treatment group
Details on study design:
CAM preparation:
The number of eggs used in the study was 3 per treatment group. The air cell of the eggs were marked and cut off with a rotating saw blade. The uncovered inner membrane was moistened with physiological saline and subsequently the egg was kept at room temperature for 1-10 minutes. After this period the physiological saline was removed and the inner egg membrane carefully eliminated using forceps to expose the CAM.
Negative control:
A negative control (0.9% (w/v) NaCl (Merck, Darmstadt, Germany) in Milli-RO water (Millipore Corp., Bedford, MA., USA) (physiological saline) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
Positive control:
The positive controls was 1% (w/v) Sodium dodecyl sulphate solution prepared in physiological saline.

Results from the three test method endpoints were evaluated separately for each egg.

The irritancy score (IS) was calculated using the formula:
IS = [((301-Haemorrhage time)/300)x5] + [((301-Lysis time)/300)x7] + [((301-Coagulation time)/300)x9]

IS = irritancy score for total effect
Haemorrhage time = observed start (in seconds) of haemorrhage reactions on CAM
Lysis time = observed start (in seconds) of vessel lysis on CAM
Coagulation time = observed start (in seconds) of coagulation formation on CAM

Data evaluation:
The mean score for each treatment was calculated from the final scores obtained for each egg in that treatment and evaluated according to the following irritancy classification scheme of Luepke (1985) and Kalweit et al. (1987).

HET-CAM Irritancy Score (IS) Range Irritation Category
0 – 0.9 Not irritant
1 – 4.9 Slight Irritant
5 – 8.9 Moderate Irritant
9 – 21 Severe Irritant

Acceptability of the assay:
The assay will be acceptable if:
- The positive controls give an in vitro irritancy score that is in range or close to the historical in vitro irritancy score mentioned in the BRD document: 1% (w/v) Sodium dodecyl sulphate: 8-14: NOTOX: mean 14.8 ± 0.4 (range: 14.1 – 15.3).
- The negative control gives an in vitro irritancy score of less than 1.

Irritation parameter:
other: in vitro irritancy score
Basis:
mean
Time point:
other: 20-300 sec
Score:
16
Reversibility:
not specified
Remarks on result:
other: mean of 3 eggs
Irritant / corrosive response data:
The coagulation was observed directly after rinsing the test substance of from the chorioallantoic membrane. The haemorrhages occurred on the treated parts of the membrane and vessel lysis on the non-treated parts of the membrane.
The mean irritancy score of the negative control was 0.
The mean irritancy score of the 1% Sodium dodecyl sulphate was 15.2.
Interpretation of results:
other: severe irritant
Remarks:
Criteria used for interpretation of results: other: Luepke (1985) and Kalweit et al. (1987)
Conclusions:
The mean in vitro irritancy score obtained with the negative control was less than 1 indicating that the negative control did not induce irritancy on the chorioallantoic membrane. The mean in vitro irritancy score of the positive control (1% (w/v) Sodium dodecyl sulphate) was within the historical control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The mean in vitro irritancy score obtained during a 280 seconds observation period after 20 seconds treatment with LiPF6 was 16.0.

Finally, it is concluded that this test is valid and that LiPF6 is a severe irritant in the Hen’s Egg Test – Chorioallantoic Membrane Test under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
Investigation in vitro found corrosive activity (subsequently confirmed by local findings on the ears of mice in a sensitisation study).

Justification for selection of eye irritation endpoint:
Investigation in vitro found severe irritating activity.

Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: highly irritating

Justification for classification or non-classification

The properties of the test substance, determined experimentally and predictable from its known (and rapidly formed) hydrolysis product HF, trigger classification under CLP regulation (1272/2008) as corrosive (Skin Corrosion 1A, H314) and severely damaging to eyes (Eye Damage 1, H318). In view of the inclusion of reference to eye damage in H314, separate labelling in respect of eye damage may not be required.