Dilithium tetraborate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: • EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dilithium tetraborate

EC Number:

234-514-3

EC Name:

Dilithium tetraborate

Cas Number:

12007-60-2

Molecular formula:

B4Li2O7

IUPAC Name:

dilithium tetraborate

Test material form:

solid: bulk

Specific details on test material used for the study:

Test item storage: At room temperature

In vitro test system

Test system:

other: EpiDerm Skin Model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

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.

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 (Dulbecco’s Modified Eagle’s Medium). The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test item was applied. The skin was moistened with 25 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and 34.3 to 42.0 mg of the solid test item was added into the 6-well plates on top of the skin tissues.

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.

After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item followed by immediate determination of the cytotoxic (corrosive) effect. 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.

For the cell viability measurements, the DMEM was replaced by 300 µl MTT-medium and the tissues were incubated for 3 hours at 37°C in air containing 5% CO2. Following incubation, the tissues were washed with PBS and formazan and then extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

In the test, 34.3 to 42.0 mg of the solid test item was added directly to the top of the skin tissues. No vehicle was used. The skin was dampened with Milli-Q water prior to the test item application.

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)

Duration of treatment / exposure:

Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.

Duration of post-treatment incubation (if applicable):

After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item followed by immediate determination of the cytotoxic (corrosive) effect. The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

Number of replicates:

The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute

Value:

93

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

not corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

not corrosive

Other effects / acceptance of results:

The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Results showed that the solutions did not turn blue / purple nor a blue / purple precipitate was observed. It was therefore concluded that the test item did not interfere with the MTT endpoint.

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 93% and 99% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

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. The mean relative tissue viability following the 1-hour exposure to the positive control was 12%.

In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was <17%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1          Mean Absorption in the in vitro Skin Corrosion Test with the test item

	3-minute application				1-hour application
	A (OD570)	B (OD570)	Mean (OD570)	SD	A (OD570)	B (OD570)	Mean (OD570)	SD
Negative control	1.773	1.920	1.847	0.104	1.958	1.616	1.787	0.242
The test item	1.822	1.629	1.725	0.136	1.608	1.937	1.772	0.233
Positive control	0.220	0.245	0.233	0.017	0.203	0.213	0.208	0.007

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0434). Isopropanol was used to measure the background absorption.

Table 2          Mean Tissue Viability in the in vitro Skin Corrosion Test with the test item

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
The test item	93	99
Positive control	13	12

Table 3          Coefficient of Variation between Tissue Replicates

	3 minute	1 hour
Negative control	7.7	17
The test item	11	17
Positive control	10	4.9

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Lithium tetraborate is not corrosive in the in vitro skin corrosion test according to OECD 431.

Executive summary:

An in vitro skin corrosion test was conducted according to OECD 431. The test item was applied directly to moistened human skin and performed on a total of 4 tissues per test item together with a negative control and positive control.  Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.  The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 93% and 99% respectively.  Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive. In addition, the acceptance criteria was met for both the positive and negative control.