Calcium tetraborate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2017 - 20 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Not applicable

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

Justification for test system used:

In the interests of animal welfare, the validated in vitro skin irritation test, EPISKIN test has been conducted initially in order to minimize the need for in vivo testing.

Vehicle:

unchanged (no vehicle)

Details on test system:

The EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 17-EKIN-039) is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 22.5 hours at 37°C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The solid test item was applied directly to the skin tissue. The skin was initially moistened with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item. The amount of test item applied ranged from 14.2 mg to 22.2 mg.

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

Duration of post-treatment incubation (if applicable):

The skin tissues were placed in 2 mL warmed maintenance medium until all tissues were dosed and rinsed. The skin tissues were then incubated for 42 hours at 37°C.

After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/ml in PBS) and then the tissues were incubated for a further 3 h at 37°C.

Number of replicates:

3 tissues per test item together with negative and positive controls.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control was determined to be 1.028 with a standard deviation of 0.052 which met the required acceptance criteria (SD<18%).

The mean relative tissue viability of the positive control was determined to be 11% with a standard deviation of 3.7 which was <50% relative to the negative control and within the Standard Deviation (SD) acceptance criteria of <18%.

The mean tissue viability for the test item was determined to be 89%. The standard deviation of the viability of the test item treated tissues was 19% which is above acceptance criteria. However, since all individual viabilities were >50%, the test outcome was considered valid.

Any other information on results incl. tables

Table 1           Mean Absorption in the In Vitro Skin Irritation Test with Dilithium tetraborate

		A (OD570)		B (OD570)		C (OD570)		Mean (OD570)				SD
Negative control		0.978		1.082		1.025		1.028		±		0.052
Test item		1.076		0.698		0.962		0.912		±		0.194
Positive control		0.090		0.161		0.102		0.118		±		0.038

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.043). Isopropanol was used to measure the background absorption.

Table 2       Mean Tissue Viability in the In VitroSkin Irritation Test with Dilithium tetraborate

		Mean tissue viability (percentage of control)		Standard deviation (percentage)
Negative control		100		5.1
Test item		89		19
Positive control		11		3.7

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, an in vitro skin irritation test was conducted according to OECD 439. Dilithium tetraborate was found to be non-irritant and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

An in vitro skin irritation test was conducted according to OECD 439. The test item was applied directly to moistened human skin for 15 minutes. After exposure, the skin was thoroughly rinsed and transferred to fresh medium. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test item.  A negative and positive control were also conducted in parallel.  

 

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Dilithium tetraborate compared to the negative control tissues was 89%. Since the mean relative tissue viability was above 50% after 15 ± 0.5 minutes treatment, Dilithium tetraborate was considered as non-irritant.

 

The positive control had a mean cell viability of 11% after 15 ± 0.5 minutes exposure.  The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 6% for the negative and positive control, indicating that the test system functioned properly.