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Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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 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
Details on test animals and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.
Vehicle:
water
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

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 %.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-08-03 to 2010-08-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
, 2002-04-24
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-11-12
Species:
rabbit
Strain:
Himalayan
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at study initiation: Approx. 4.5 - 5.5 months
- Weight at study initiation: Animal 1: 2.5 kg; Animal 2: 2.4 kg; Animal 3: 3.1 kg
- Housing: For 8 hours following test item application, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn, wiping of the eyes with the paws and excluded irritation of the eye by excrements and urine. During the acclimatisation period and after the 8-hour period in restrainers, the animals were kept singly in cages with dimensions of 380 mm x 425 mm x 600 mm (manufacturer: Dipl.Ing. W. EHRET GmbH, 16352 Schönwalde, Germany).
- Diet (ad libitum): Commercial diet, ssniff7 K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum before and after the exposure period): Tap water
- Acclimation period: At least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C +/- 3 °C (maximum range)
- Relative humidity: 30% - 70% (maximum range; aim was 50% - 60%)
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg of the test item were administered into one eye each of three animals. The test item was placed into the conjunctival sac of the right eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the material. The left eye, which remained untreated, served as a control.
No further information on the amount/concentration applied was stated.
Duration of treatment / exposure:
1 hour (One hour after application the eyes were rinsed.)
Observation period (in vivo):
1, 24, 48 and 72 hours after the administration
Number of animals or in vitro replicates:
3 male rabbits
Details on study design:
The test was performed initially using one animal. As no corrosive or severe irritant effects were observed in this animal, 2 further animals were employed 24 hours after start of the initial test.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The eyes were rinsed with portions of 20 mL 0.9 % aqueous NaCl solution, each.
- Time after start of exposure: 1 hour after administration

SCORING SYSTEM: draize scoring system

TOOL USED TO ASSESS SCORE: The eyes were examined ophthalmoscopically with a slit lamp prior to the administration and 1, 24, 48 and 72 hours after the administration. The eye reactions were observed and registered.
24 hours after administration, fluorescein (Fluorescein SE Thilo drops (ALCON PHARMA GmbH, 79108 Freiburg, Germany) was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions.

OTHER OBSERVATIONS: Any further lesions not covered by the scoring system were recorded. Body weight of all animals was measured at the beginning of the study and at the end of the study. Behaviour and food consumption were monitored.
No further information on the study design was stated.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritant / corrosive response data:
Conjunctival redness (grade 1 (Some blood vessels hyperaemic (injected)) was observed in all animals 1 hour after instillation. This effect was reversible after 24 hours.
In addition, secretion was observed in all animals 1 hour after instillation.
The cornea and the irises were not affected by instillation of the test item and the fluorescein test performed 24 hours after instillation did not reveal any changes.
Other effects:
There were no systemic intolerance reactions.
Interpretation of results:
GHS criteria not met
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the EC-Commission directive 67/548/EEC and its subsequent amendments on the approximation of the laws, regulations and administrative provision relating to the classification, packaging and labelling of dangerous substances and the results obtained under the present test conditions barium carbonate - standard was non-irritating to eyes, hence, no labelling is required.
According to the EC Regulation 1272/2008 and subsequent regulations, the test item is non-irritating to eyes; no classification and labelling of the substance is necessary.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Reason for read across from BaCl2to BaCO3:

The toxicity of barium carbonate and barium chloride may reasonably be considered to be determined by availability of Ba2+cations. As a first surrogate for bioavailability, the water solubility of a test substance may be used. Barium carbonate is poorly soluble in water whereas barium chloride is well water soluble.Read-across from soluble to the poorly soluble barium compound is considered acceptable because toxicokinetic data on the poorly soluble barium carbonate indicates a markedly lesser bioavailability than the soluble barium chloride. However, it is to be concluded that this read-across will likely lead to rather conservative no-effect levels.

Skin irritation:

The in vitro study according to the draft EC method B.46 (EpiSkinTM) was validated and considered of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, and for being used as a replacement of in vivo method OECD 404 for the purposes of distinguishing between R38 skin irritating and non-skin irritating test substances (ECVAM, 27.04.2007).

Justification for classification or non-classification

Skin irritation:

Reference Verbaan (2010) is considered as the key studies for in vitro skin irritation and will be used for classification. The overall irritation result is as follows: Relative viability: mean after 15 min incubation 80% (max. score: 88%); evaluated by MTT reduction. The classification criteria according to regulation (EC) 1272/2008 as irritating to skin are not met since the mean rel. viability is >50% of the control, hence no classification required.

 

Eye irritation:

Reference Leuschner (2010) is considered as the key study for eye irritation and will be used for classification. The overall irritation results are as follows: Corneal opacity, iritis, chemosis and conjunctivae, 24, 48 and 72h after application: max score=0

The classification criteria according to regulation (EC) 1272/2008 as irritating to eyes are not met, no classification and labelling for barium carbonate is required.

 

Respiratory irritation:

The classification as respiratory irritant is covered under the endpoint specific target organ toxicity- single exposure and repeated exposure. Please refer to the endpoint summaries on acute toxicity (endpoint 7.2) and repeated dose toxicity (endpoint 7.5) for further information.