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Toxicological information

Skin irritation / corrosion

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
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

Constituent 1
Chemical structure
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(1+) bicyclo[3.3.1]tetraboroxane-3,7-bis(olate)
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

Resultsopen allclose all
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
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.