Lithium [ethanedioato-O,O’]tetrafluorophosphate

Endpoint summary

Description of key information

In vitro skin corrosion:

The ability of test item to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)) was assessed based on most recent OECD 431. The test item is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

BCOP test:

The eye hazard potential of test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea was assessed using the Bovine Corneal Opacity and Permeability test (BCOP test) based on the most recent OECD guideline 437.

The test item induced an IVIS > 55 (actual value: 180), it is concluded that test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

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:

From 2018-12-17 to 2019-03-08

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:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch No.: 180410
Purity: 99%

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): EPI-200, Lot no.: 29674 kit K and L, 29944 kit I and J, 29987 kit C and D

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 36.3– 37.6°C for 1-hour exposure; room temperature for 3-minutes exposure
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues (= one application time) were dosed and rinsed.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm


NUMBER OF REPLICATE TISSUES:
Two tissues were used for a 3-minute exposure, two for a 1-hour exposure. For the negative and positive controls, 2 tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues were treated with 50 μL 8N KOH (positive control) for both the 3-minute and 1-hour time point.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): In the first experiment: 36.41 to 95.16 mg; In the second and third experiment an excessive amount of the test item was applied, so that the skin tissue was completely covered

NEGATIVE CONTROL
- Amount(s) applied: 50 μL

POSITIVE CONTROL
- Amount(s) applied: 50 μL
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3-minute exposure and 1-hour exposure

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute treatments

Value:

>= 32 - <= 38

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour treatments

Value:

>= 0.6 - <= 4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction and Colour interference with MTT: 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 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
- Acceptance criteria met for positive control: The mean relative tissue viability following the 1-hour exposure to the positive control was 6.6, 13 and 7.1%, respectively.
- Acceptance criteria met for variability between replicate measurements: In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was
≤ 19% for the negative control and test item. Except for tissues treated for 3 minutes with test item in the first two experiments, which had a Coefficient of Variation of 90%, but therefore these experiments were repeated. For the positive control in experiment 2, the Coefficient of Variation between tissue replicates at the 3-minute exposure was 36%. Since both individual tissues were clearly positive it was concluded that the test system functioned properly.

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

In conclusion, the test item is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

The ability of test item to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)) was assessed based on most recent OECD 431. The possible corrosive potential of test item was tested through topical application for 3 minutes and 1 hour.

Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of test item was applied directly on top of the skin tissue. In total, 3 experiments were performed. In the first two experiments, after 3 minute exposure to the test item, the individual results of the two tissues were spread over 2 categories, with a subsequent high coefficient of variation, and therefore these experiments were repeated.

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with test item compared to the negative control tissues was 32% and 4.0%, respectively in the first experiment, 38% and 3.6%, respectively in the second experiment, and 41% and 0.6%, respectively in the third experiment. Because the mean relative tissue viability for test item was below 15% after the 1-hour treatment in all three experiments, it is considered to be corrosive.

In conclusion, test item 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:

2018-12-04

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:

2017

Deviations:

yes

Remarks:

In the test item treated group, one cornea out of three was excluded from measurement as it was punctured. As other two measurements showed comparable results, this deviation had no influence on the study results.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch No.: 180410
Purity: 99%

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Vitelco, -'s Hertogenbosch, The Netherlands
- 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:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): not specified

Duration of treatment / exposure:

240 ± 10 minutes

Number of animals or in vitro replicates:

3 (1 was excluded from measurement)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

NUMBER OF REPLICATES : 3 each for negative, positive control and test item

NEGATIVE CONTROL USED : physiological saline

POSITIVE CONTROL USED : 20% (w/v) Imidazole solution

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750 μL of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others: Possible pHeffects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Value:

180

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
The corneas treated with the test item showed opacity values ranging from 168 to 191 and permeability values ranging from 0.020 to 0.076. The corneas were turbid after the 240 minutes of treatment with the test item. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 169 to 192 after 240 minutes of treatment with test item.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 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.
- Acceptance criteria met for positive control: The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 124 and within two standard deviations of the current historical positive control mean.

Interpretation of results:

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

Conclusions:

In conclusion, since test item induced an IVIS > 55 (actual: 180), it is concluded that test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Executive summary:

The eye hazard potential of test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea was assessed using the Bovine Corneal Opacity and Permeability test (BCOP test) based on the most recent OECD guideline 437.

The eye damage of test item was tested through topical application to isolated bovine corneas for approximately 240 minutes.

The test item induced mainly serious opacity eye damage, resulting in a mean in vitro irritancy score of 180 after 4 hours of treatment. In conclusion, since test item induced an IVIS > 55, it is concluded that test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin corrosion in vitro: OECD 431, corrosive

According to Regulation (EC) No 1272/2008, section 3.2.6 and Table 3.2.1, this substance should be classified as category 1 for this endpoint.

Eye irritation in vitro: OECD 437: Positive, serious eye damage

According to Regulation (EC) No 1272/2008, section 3.3.2 and Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) Figure 3.3.1, this substance should be classified as category 1 for this endpoint.