Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-04-26 to 2010-06-07
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Draft Proposal for a New Guideline: In vitro skin irritation: Reconstructed Human Epidermis (RhE) Test method
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission regulation (EC) No. 440/2008 B.46
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Barium chloride di-hydrate
- EC number: 233-788-1
- Molecular formula: BaCl2 * 2H2O
- Molecular weight: 244.26
- Physical state: White crystalline powder (determined at NOTOX)
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- pH: 5.5 - 7.5 (100 g/L at 20 °C)
- Stability in water: not indicated
Solubility in water 400 g/L (20 °C)
No further information on the test material was stated

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: Adult human-derived epidermal keratinocytes
Cell source:
other: three-dimensional human epidermis model
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing and therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
EPISKIN Standard Model TM (EPISKIN-SM TM, 0.38 cm2, Lot no.: 10-EKIN-020; Source: SkinEthic Laboratories, Nice, France).
This model 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.

TEST FOR REDUCTION OF MTT BY THE TEST SUBSTANCE
Barium chloride was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, approximately 10 mg of test substance was added to a 12 well plate filled with 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for approximately 3 hours at 37 °C. A negative control, sterile Milli-Q water was tested concurrently. Because no colour change was observed it was concluded that barium chloride dihydrate did not interact with MTT.

APPLICATION/TREATMENT OF THE TEST SUBSTANCE
The test was performed on a total of 3 tissues per test substance together with a negative control (Phosphate buffered saline (PBS, Invitrogen Corporation, Breda, the Netherlands) and a positive control (5% (aq) Sodium dodecyl sulphate (SDS, Sigma Aldrich, Zwijndrecht, The Netherlands, CAS Number 151-21-3). At least 10 mg solid (with a small glass weight boat) with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., U.S.A.) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 10 µl PBS (negative control) and 3 tissues with 10 µl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully. The skin tissues were kept in new 12-well plates on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37 °C.

CELL VIABILITY MEASUREMENT
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-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37 °C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Multiskan Spectrum (Thermo Labsystems).
Cell viablity was calcuated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): At least 10 mg of the solid test substance was applied directly on top of the skin tissue. Barium chloride was spread to match the size of the tissue.

VEHICLE
- Amount(s) applied (volume or weight with unit): Skin tissue was moistened with 5µl of Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact to the tissue.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 tissues

Test animals

Details on test animals and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:
water

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
80
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

TEST FOR REDUCTION OF MTT BY THE TEST SUBSTANCE

Because no colour change was observed it was concluded that barium chloride dihydrate did not interact with MTT.

Results after treatment with test substance:

Table 1. Individual OD measurements at 570 nm

 

A

(OD570)

B

(OD570)

C

(OD570)

Negative control

OD570measurement 1

OD570measurement 2

 

0.901

0.877

 

0.884

0.875

 

0.897

0.870

Barium chloride dihydrate

OD570measurement 1

OD570measurement 2

 

0.734

0.574

 

0.733

0.738

 

0.747

0.738

Positive control

OD570measurement 1

OD570measurement 2

 

0.050

0.056

 

0.061

0.063

 

0.069

0.067

OD = Optical density

Triplicate exposures are indicated by A, B and C.

The mean absorption at 570 nm measured after treatment with barium chloride dihydrate and controls are presented in Table 2.

Table 2.  Mean absorption in the in vitro skin irritation test with barium chloride dihydrate 

 

A

(OD570)

B

(OD570)

C

(OD570)

Mean

(OD570)

SD

Negative control

0.889

0.879

0.883

0.884

0.005

Barium chloride dihydrate

0.654

0.735

0.742

0.711

0.049

Positive control

0.053

0.062

0.068

0.061

0.008

 

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. Isopropanol was used to measure the background absorption.

 

Table 3 shows the mean tissue viability obtained after 15 minutes treatment with barium chloride dihydrate compared to the negative control tissue.

Table 3. Mean tissue viability in the in vitro skin irritation test with barium chloride dihydrate 

 

Mean tissue viability (percentage of control)

Negative control

100

Barium chloride dihydrate

80

Positive control

7

The positive control had a mean cell viability after 15 minutes exposure of 7 %.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
It is concluded that this test is valid and that barium chloride dihydrate is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

An in vitro skin irritation test of Barium chloride was performed in a reconstructed human epidermis model EPISKIN (SM). Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The 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 in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with Barium carbonate, the mean cell viability was 80% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.