Trilithium orthophosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-08 to 2015-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Trilithium orthophosphate

EC Number:

233-823-0

EC Name:

Trilithium orthophosphate

Cas Number:

10377-52-3

Molecular formula:

Li3O4P

IUPAC Name:

trilithium orthophosphate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch No.of test material: 1153
- Expiration date of the lot/batch: May 2020

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

Test animals

Species:

other: EpiSkin™ SM kit

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- Source: EPISKIN SNC Lyon, France

Test system

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).

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of three tissues

Value:

89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

test substance, mean OD570: 0.614

Any other information on results incl. tables

Table 1: OD values and viability percentages

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1 x PBS	1	0.751	108
	2	0.680	98
	3	0.648	93
	mean	0.693	100
	standard deviation (SD)		7.65
Positive control: SDS (5% aq.)	1	0.056	8
	2	0.070	10
	2	0.034	5
	mean	0.053	8
	standard deviation (SD)		2.59
Test item	1	0.555	80
	2	0.624	90
	3	0.663	96
	mean	0.614	89
	standard deviation (SD)		7.90

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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

According to the available in vitro test, lithium phosphate 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 1 x 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 less than or equal to (≤) 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 phosphate 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 phosphate is considered as non-irritant to skin and is therefore not classified.