Lithium acetate

Endpoint summary

Description of key information

According to the two in vitro tests (Corrositex and EPISKIN), lithium acetate is not regarded as irritating to the skin.

Under the conditions of the test system, lithium acetate is considered as eye irritant in the EpiOcular™ Eye Irritation Test (Cat. 2 , H319).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-07-08 to 2015-07-10

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)

Version / remarks:

(2013)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 09406DE

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Expiration date of the lot/batch: May 2018
- Storage condition of test material: Store at room temperature (15-30 °C). Keep container tightly closed in a dry and well-ventilated place.

Species:

other: EpiSkin™ SM kit

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- Source: EPISKIN SNC Lyon, France

Vehicle:

unchanged (no vehicle)

Controls:

other: Concurrent treatment with 1x PBS (negative control) and 5% aq. SDS (postive control)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature.

Observation period:

After rinsing, the units were placed into the plate wells filled with fresh pre-warmed “Maintenance Medium” (2 mL/well) below them and then incubated for 42 hours at 37 °C in an incubator with 5 % CO2. After the 42 hours incubation the skin units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours at 37 °C in an incubator with 5 % CO2 protected from light.

Details on study design:

PERFORMANCE OF THE STUDY
Pre-incubation (day -1)
The “maintenance medium” was pre-warmed to 37 °C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed above the media in a separately prepared well. Contact of the epidermis units with the media was assured. The well was then incubated overnight at 37 °C in an incubator with 5 % CO2.

Application, Exposure and Rinsing (day 0)
Test Item
The epidermal surface was first moistened with 5 μL deionised water* (in order to improve further contact between powder and epidermis) and then 10 mg of the test item was applied evenly onto the skin. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.
* prepared by MILLIPORE ELIX 3 water purification system in TOXI-COOP ZRT.
Positive and negative controls
A volume of 10 μL positive control (SDS 5 % aq.) or negative control (1 x PBS) was applied to the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature.

After the incubation time, the EpiSkinSM units were removed and rinsed thoroughly with PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis).

Post-incubation (day 0-2)
After rinsing the units were placed into the plate wells filled with fresh pre-warmed “Maintenance Medium” (2 mL/well) below them and then incubated for 42 hours at 37 °C in an incubator with 5 % CO2.

MTT test (day 2)
After the 42 hours incubation the EpiSkinSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours at 37 °C in an incubator with 5 % CO2 protected from light.

Formazan Extraction (day 2)
At the end of incubation with MTT a formazan extraction step was undertaken:
A defined disk of epidermis from each replicate was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 μL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a Vortex Mixer to achieve a good contact of all of the material to the acidified isopropanol. Then the mixture was incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements
Following the formazan extraction, 2 × 200 μL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD’s of each well were recorded using a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at a wavelength of 570 nm while using an acidified isopropanol solution blank (6 × 200 μL).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test substance

Value:

90

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

(OD570 = 0.627)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative control

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

(OD570 = 0.693)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control

Value:

8

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

(OD570 = 0.053)

Other effects / acceptance of results:

All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to skin.

Interpretation of results:

GHS criteria not met

Conclusions:

According to the available in vitro test, lithium acetate is considered as non-irritant to skin.

Executive summary:

An in vitro skin irritation test was conducted according to OECD guideline 439 and EU method B.46. Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is <= 50 % when compared to the viability values obtained from the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item Lithium acetate did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 90 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions. According to the current OECD Guideline No. 439, lithium acetate is considered as non-irritant to skin and is therefore not classified.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2000-02-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

CorrositexTM is a quantitative in vitro corrosivity test. The model is based on the time required for the test substance to pass through a biobarrier membrane and produce a change in a chemical detection system.

GLP compliance:

not specified

Species:

other: not applicable

Details on study design:

The international Corrositex assay kit is an in vitro method for determining the corrosive potential of chemical substances. The results of this assay were used to determine the United Nations Transport Regulations Packaging Group. For detailed test procedure see Sect. "Any other information on materials and methods incl. tables"

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

mean of breakthrough time (min) of 4 replicates

Value:

> 60

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

One run has been performed with 4 replicates. Each run was longer than > 60 minutes and so is the mean time of the 4 replicates.

Based on the results of the Corrositex TM Assay, the test article, lithium acetate dihydrate has not to be classified as corrosive according to the UN Dangerous Goods Transport Regulations.

 

Interpretation of results:

GHS criteria not met

Conclusions:

Lithium acetate dihydrate was tested for corrosive properties in the CorrositexTM Assay which is used for assigning the packaging group according to UN Dangerous Goods Transport Regulations. Lithium acetate dihydrate was found in this test to be not corrosive.

Executive summary:

Lithium acetate dihydrate was tested for corrosive properties in the CorrositexTM Assay in a single trial (four replicates) that examines the mean breakthrough time in order to determine the packing group classification. The mean breakthrough time of the 4 replicates was > 60 minutes. Therefore, based on the evaluation of the test results, lithium acetate dihydrate was not considered to be corrosive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

(2013)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.: 09406DE

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Expiration date of the lot/batch: May 2018
- Storage condition of test material: Store at room temperature (15-30 °C). Keep container tightly closed in a dry and well-ventilated place.

Species:

chicken

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129., Hungary

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.03 g

Duration of treatment / exposure:

The gentle rinsing with 20 mL saline was performed in all test item treated eyes after the 30, 75 and 120 minutes of observation. At 180 minutes of observation one of the three eyes was totally clear, with the two other eyes gentle rising was performed again, but they were not totally cleared at 240 minutes after the post-treatment rinse.

Observation period (in vivo):

Please refer to duration of treatment.

Number of animals or in vitro replicates:

3 eyes per treatment group were used.

Details on study design:

EXPERIMENTAL PROCEDURE
Base line assessments
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

Test item treatment
After the zero reference measurements, one out of three eyes in the treatment group was held in a horizontal position and lithium acetate was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test substance, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated with the remaining two eyes in the treatment group.
The three positive control eyes were treated in a similar way with 0.03 g Imidazole.
One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 μL from micropipette, in such a way that the entire surface of the cornea was covered with negative control, taking care not to damage or touch the cornea with the application equipment.

Test item removal
The Imidazole and test item were stuck on the corneas surface in all eyes at 30, 75, and 120 minutes after the post-treatment rinse.
The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation, but cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse.
The gentle rinsing with 20 mL saline was performed in all test item treated eyes after the 30, 75 and 120 minutes of observation. At 180 minutes of observation one of the three eyes was totally clear, with the two other eyes gentle rising was performed again, but they were not totally cleared at 240 minutes after the post-treatment rinse.

Measurements
The control and test item treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.
The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

Evaluation
The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium).
Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an Isolated Chicken Eye (ICE) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance.

Irritation parameter:

percent corneal swelling

Remarks:

at up to 75 min

Run / experiment:

three eyes tested

Value:

1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I (test substance)

Irritation parameter:

percent corneal swelling

Remarks:

at up to 240 min

Run / experiment:

three eyes tested

Value:

2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I (test substance)

Irritation parameter:

other: Mean maximum corneal opacity

Run / experiment:

three eyes tested

Value:

0.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II (test substance)

Irritation parameter:

other: Mean fluorescein retention

Run / experiment:

three eyes tested

Value:

2.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class IV (test substance)

Irritation parameter:

other: Overall ICE class

Run / experiment:

three eyes tested

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 1xI, 1xII, 1xIV (test substance)

Irritation parameter:

percent corneal swelling

Remarks:

at up to 75 min

Run / experiment:

three eyes tested

Value:

25

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class III for positive control: imidazole

Irritation parameter:

percent corneal swelling

Remarks:

at up to 240 min

Run / experiment:

three eyes tested

Value:

34

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class IV for positive control: imidazole

Irritation parameter:

other: mean maximum corneal opacity

Run / experiment:

three eyes tested

Value:

4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class IV for positive control: imidazole

Irritation parameter:

other: mean fluorescein retention

Run / experiment:

three eyes tested

Value:

3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class IV for positive control: imidazole

Irritation parameter:

other: Overal ICE Class

Run / experiment:

three eyes tested

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 3xIV for positive control imidazole

Irritation parameter:

percent corneal swelling

Remarks:

at up to 75 min

Run / experiment:

one eye tested

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I for negative control: saline solution

Irritation parameter:

percent corneal swelling

Remarks:

at up to 240 min

Run / experiment:

one eye tested

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I for negative control: saline solution

Irritation parameter:

other: mean maximum corneal opacity

Run / experiment:

one eye tested

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I for negative control: saline solution

Irritation parameter:

other: mean fluorescein retention

Run / experiment:

one eye tested

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I for negative control: saline solution

Irritation parameter:

other: Overal ICE Class

Run / experiment:

one eye tested

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 3xI for negative control: saline solution

Irritant / corrosive response data:

Ocular corrosion and severe irritation potential were not observed for the test substance. Based on the overall ICE Class the positive control Imidazole was classified as corrosive/severely irritating, UN GHS Classification: Category 1. Based on the overall ICE Class the negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

Interpretation of test results

Table 4 Overall in vitro irritancy classifications:

 

UN GHS Classification	Combinations of the 3 Endpoints
No Category	3xI, 2xI, 1xII
No prediction can be made	Other combinations
Category 1 (Corrosive/Severe Irritant)	3×IV 2×IV, 1×III 2×IV, 1×II^1 2×IV, 1×I^1 Corneal opacity >= 3 at 30 min (in at least 2 eyes) Corneal opacity = 4 at any time point (in at least 2 eyes) Severe loosening of the epithelium (in at least 1 eye)

 

Remark:

¹: combinations of categories less likely to occur

 

Note:  Small negative numbers for swelling following application are counted as zero (larger negative numbers due to erosion invalidate the swelling evaluation, but indicate a severe effect (scored as class IV)).

Interpretation of results:

other: No prediction can be made

Conclusions:

Based on the results of the in vitro test for eye corrosives and severe irritants in isolated chicken eyes, ocular corrosion and severe irritation potential was not observed for the test substance. On the other hand, the results were not conclusive regarding differentiation between irritation or non irritation potential.

Executive summary:

In order to evaluate the potential ocular corrosivity and irritancy of the test item lithium acetate an isolated chicken eye test was performed according to OECD guideline 438 and EU method B.48. The test item lithium acetate and positive control (Imidazole) were ground before use in the study. They were applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, lithium acetate did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. The experiments were considered to be valid.

In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with lithium acetate, no ocular corrosion or severe irritation potential was observed. According to guideline OECD 438, lithium acetate overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, test item has been categorized as “No prediction can be made”.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-07-11 to 2016-07-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28th July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

LAUS GmbH, Auf der Schafweide 20, 67489 Kirrweiler, Germany

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 09406DE
- Expiration date of the lot/batch: May 2018


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15-30°C) Keep containers tightly closed in a dry and well-ventilated place

Species:

human

Strain:

other: three-dimensional non-keratinized tissue construct

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied:
Tissue 1: 51.2 mg
Tissue 2: 51.7 mg

Duration of treatment / exposure:

5 hours and 53 minutes

Duration of post- treatment incubation (in vitro):

17 hours and 58 minutes

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
Preparations: On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the MTT solvent and the solution was stored at 2 - 8 °C in the dark.
The assay medium was warmed in the water bath to 37 ± 1°C. 6-well-plates were labelled with test item, resp. negative control, resp. positive control and filled with 1 mL assay medium in the appropriate wells. All 24 inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 - 100 % relative humidity for 55 minutes. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 -100 % relative humidity for 16 hours (16 - 24 hours).

Exposition and Post-Treatment: After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 - 100 % relative humidity for 30 minutes. After that, 50 µL of the controls and a defined amount of the test item (Tissue 1: 51.2 mg; Tissue 2: 51.7 mg) were applied in duplicate in one-minute-intervals. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 5 hours and 53 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 - 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature.
After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 17 hours and 58 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 - 100 % relative humidity. After the post-treatment incubation, the MTT Assay was performed.

- RhCE tissue construct used, including batch number:
EpiOcular™ tissues were produced from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Day of delivery: 12th July 2016
Batch no.: 23719
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
5 hours and 53 minutes at 37 ± 1 °C; 25 minutes post soak at room temperature; 17 hours and 58 minutes at 37 ± 1 °C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
The Pre-Test showed, that the substance does not interact directly with MTT. Thus, additional controls were not necessary.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength and measuring device: 570 nm; plate spectral photometer
- Description of the method used to quantify MTT formazan
A 24-well-plate was prepared with 300 µL freshly prepared MTT-reagent in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 175 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 - 100 % relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature.
The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate. Eight wells with 200 µL isopropanol were additionally pipetted. The plate was read in a plate spectral photometer at 570 nm.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The values of the 96-plate-reade were transferred into a spreadsheet (Microsoft Excel®). Aoll calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly
The Eye irritation is assessed using the following criteria (source: MatTek Corporation)
- the test item is considered as Non Eye Irritant if the mean percent tissue viability is greater than 60%
- the test item is considered as Eye Irritant (GHS category 1 or 3) if the mean percent tissue viability is smaller than or equal to 60%
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Historical positive and negative control data are provided by the conducting laboratory.
- Complete supporting information for the specific RhCE tissue construct used: Certificate of Analysis on test system quality is provided by the supplier (MatTek Corporation)
- Positive and negative control means and acceptance ranges based on historical data
Acceptance range negative control optical density (OD): ≥ 0.8 and ≤ 2.5
Acceptance range positive control in percent Formazan production: < 50% of negative control
- Acceptable variability between tissue replicates for positive and negative controls: < 20%
- Acceptable variability between tissue replicates for the test chemical: < 20%

Irritation parameter:

other: Tissue viability (%)

Run / experiment:

Mean of 2 replicates

Value:

3.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The OD value was 1.6 (>= 0.8 and <= 2.5)
- Acceptance criteria met for positive control: Yes. The positive control reduced a decrease in the relative absorbance as compared to the negative control to 23.9 %. Variation within the replicates was acceptable (< 20 %).

Table 1: Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.036	0.035	0.034	0.036	0.035	0.036	0.035	0.036	0.035

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in table 1.

Table 2: Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Positive Control	Lithium acetate
Tissue 1	1	1.658	0.419	0.090
	2	1.771	0.443	0.087
Tissue 2	1	1.509	0.402	0.089
	2	1.674	0.421	0.088

From the measured absorbance, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in table 1 (=corrected values).

Table 3: Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	Lithium acetate
Mean – blank (Tissue 1)	1.680	0.396	0.054
Mean – blank (Tissue 2)	1.557	0.377	0.054

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the conditions of the test system, lithium acetate is considered as eye irritant in the EpiOcular™ Eye Irritation Test.

Executive summary:

In order to evaluate the potential of lithium acetate to evoke eye irritation an EpiOcular™ Eye Irritation Test (EIT) according to OECD guideline 492 was conducted. The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 5 hours and 53 minutes. After treamtent, the respective substance was rinsed from the tissue; then, cell viability of the tissue was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control, methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 <= mean OD <= 2.5, OD was 1.6. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 23.9 % (< 50%). Variation within tissue replicates was acceptable (< 20 %). After treatment with the test item, the relative absorbance value were reduced to 3.3%. This value is well below the threshold for eye irritation potential (<= 60 %).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

 

EPISKIN assay

An in vitro skin irritation test was conducted according to OECD guideline 439 and EU method B.46. Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. SDS 5 % aq. and 1 × PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is <= 50 % when compared to the viability values obtained from the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item lithium acetate did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 90 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. (TOXICOOP, 2015)

 

Corrositex Assay

Lithium acetate × 4 H2O was tested for corrosive properties in the Corrositex TM Assay in a single trial (four replicates) that examines the mean breakthrough time in order to determine the packing group classification. The mean breakthrough time of the 4 replicates was > 60 minutes, therefore, based on the evaluation of the test results, the substance was not considered to be corrosive (FMC, 2001).

 

Eye irritation/corrosion

ICE assay

In order to evaluate the potential ocular corrosivity and irritancy of the test item lithium acetate an isolated chicken eye test was performed according to OECD guideline 438 and EU method B.48. The test item lithium acetate and positive control (Imidazole) were ground before use in the study. They were applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, lithium acetate did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. The experiments were considered to be valid.

In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with lithium acetate, no ocular corrosion or severe irritation potential was observed. According to guideline OECD 438, lithium acetate overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, test item has been categorized as “No prediction can be made”.

The test method does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed. (TOXICOOP, 2015) .

RhCE assay

For the further assessment of the eye irritation potential of the test substance the EpiOcular™ Eye Irritation Test (EIT) according to OECD guideline 492 was conducted.

The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 5 hours and 53 minutes. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissue was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control, methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 <= mean OD <= 2.5, as the OD was 1.6. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 23.9 % (< 50 %). Variation within tissue replicates was acceptable (< 20 %). After treatment with the test item, the relative absorbance values were reduced to 3.3 %. This value is well below the threshold for eye irritation potential (<= 60 %). Under the conditions of the test system, lithium acetate is considered as eye irritant in the EpiOcular™ Eye Irritation Test (Cat. 2 , H319).

CONCLUSION

According to the two in vitro tests (Corrositex and EPISKIN), lithium acetate is not regarded as irritating to the skin. According to the OECD guideline study 492, lithium acetate has to be classified as Cat. 2 (H319).

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance does not need to be classified and labelled as skin irritant but needs to be classified as Cat. 2 (H319: Causes serious eye irritation) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.