Barium chloride

Endpoint summary

Description of key information

An in vitro skin irritation study (EpiSkinTM), and in vivo skin irritation study (OECD 404) and an in vivo eye irritation study (OECD 405) were performed with barium dichloride dihydrate, which indicated that the test substance is not irritating to the skin but irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 Jan 2021 (Study Plan) to 23 Feb 2021 (Final Report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This in vivo study is required by regulatory authorities for registration in a non EU region. An in vitro skin irritation study in the EPISKIN model performed by NOTOX B.V. (Project 493070) showed that the test item was non-irritant to skin and confirms that the test item is non-corrosive. The pH of the test item was determined at the Pharmacy of Charles River Laboratories Hungary Kft. and found to be 5.38. This was within the acceptable range given by the OECD guideline (If the pH is 2 or less or 11.5 or greater, the study will not be conducted), therefore the test item was permitted to use in this study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Source: S&K-LAP Kft. 2173 Kartal, Császár út 135, Hungary
- Age at study initiation: 11 weeks
- Weight at study initiation: body weights were 2907-3008 g at dosing
- Housing: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Dumbbell Device were used as an additional enrichment.
- Diet: Standard diet for rabbits produced by Cargill Takarmány Zrt, ad libitum
- Water: Free access to tap water.
- Acclimation period: 11 days before start of treatment under the same controlled environment conditions as during the experimental period. At least prior to dosing, it was ensured that the animals were healthy and that the skin to be treated was intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature: 18.2 - 22.6°C (target: 20 ± 3°C
- Humidity: 48 - 69% (target 30 - 70%)
- Air changes: 15-20 air exchanges/hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 02 Feb 2021 To: 23 Feb 2021.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
Amount(s) applied: An amount of 0.5 g of the test item (pulverised) was applied. The test item was moistened with an appropriate amount of water, sufficient to ensure good contact with the skin using a syringe.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3 males

Details on study design:

TEST SITE
Approximately 24 hours prior to the test the hair was clipped from the back and flanks of the animals.The test item was applied to an area of approximately 6 cm² of intact skin. Sterile gauze pads were placed on the skin of rabbits. These gauze pads were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The entire trunk of the animals were then wrapped with plastic wrap held in place with an elastic stocking for 4 hours.

REMOVAL OF TEST SUBSTANCE
Four hours after application, the bandaging was removed and the any residual test item was removed by gently washing the skin with body temperature water.

OBSERVATION TIME POINTS
For ethical reasons, a single animal was treated with the test item first. One hour after application of the test item to the skin, the application site was examined. As neither a corrosive effect nor a severe irritant effect was observed at one hour, the bandage was replaced and the exposure was continued for a further 3 hours (a total 4-hour exposure). The initial animal was observed for signs of erythema and oedema immediately after patch removal. The test was completed using the 2 remaining animals.

Mortality/Viability: observation twice daily (at the beginning and end of each working day).
Clinical observations: any clinical signs of toxicity or signs of ill-health during the study was recorded daily.
Body Weights: body weights were recorded at the beginning and at the end of the observation period.
Irritation: Animals were examined for signs or effects of the test item. The degree of irritation was evaluated at 1 (± 5 min), 24 (± 30 min), 48 and 72 hours (± 60 min) following the patch removal. No sign of irritation was observed at 72 hours; therefore, the study was terminated. The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

SCORING SYSTEM:

Erythema and eschar formation:
No erythema ......................................................................................................................0
Very slight erythema (barely perceptible) ............................................................................. 1
Well defined erythema ....................................................................................................... 2
Moderate to severe erythema .............................................................................................. 3
Severe erythema (beef redness) to slight eschar formation (injuries in depth) ........................... 4

Oedema formation:
No oedema ..........................................................................................................................0
Very slight oedema (barely perceptible) ................................................................................. 1
Slight oedema (edges of area well-defined by definite raising) .................................................. 2
Moderate oedema (raised approximately 1 millimeter) ............................................................ 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure)........ 4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

all the 3 animals showed the same score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Remarks:

first reading at 1h

Irritation parameter:

edema score

Basis:

mean

Remarks:

all the 3 animals showed the same score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Remarks:

first reading at 1h

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Irritant / corrosive response data:

Irritation: No skin irritation was caused by 4 hours exposure to Barium chloride dihydrate
Corrosion: There was no evidence of a corrosive effect on the skin.
Toxicity / Mortality: No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Other effects:

No histopathology was performed.

Deviations:

There were two protocol deviations in the study. The title of 18.1. Justification of Test System and Number of Eyes section in the Study Plan was mistyped, the correct title is: Justification of Test System.

The initial animal was observed for signs of erythema and oedema immediately after patch removal; however, this activity was not mentioned in the Study Plan.

Interpretation of results:

GHS criteria not met

Conclusions:

Barium chloride dihydrate does not have to be classified for skin irritation according to the CLP and the UN GHS.

Executive summary:

The primary skin irritation/corrosion of Barium chloride dihydrate in the rabbit (4-hour semi-occlusive application) was investigated in a study performed according to the OECD Testing Guideline No.404 and under GLP.

Three young adult New Zealand rabbits were treated by topical application of 0.5 g test item to their intact shaved dorsal area. The duration of treatment was 4 hours. The scoring of skin reactions was performed at approximately 1, 24, 48 and 72 hours after removal of the dressing. The primary irritation index was calculated by totalling the mean cumulative scores at 24, 48 and 72 hours and then dividing by the number of data points.

No mortality occurred during the study and no clinical signs were observed. No local dermal signs were observed in the treated animals throughout the study and the primary irritation index was 0.00. The body weights of all rabbits were considered to be within the normal range of variability. As no clinical signs were observed at 72 hours after patch removal, the study was terminated after 72 hours of observation.

Under the conditions of this study barium chloride dihydrate is considered to be "non-irritant" to rabbit skin.

Reference 2

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Qualifier:

according to guideline

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

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 or test system 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 to the test substance, 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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Skin irritation:

The primary skin irritation/corrosion of barium chloride in the rabbit (4-hour semi-occlusive application) was investigated in a study performed according to the OECD Testing Guideline No.404 and under GLP (Nagy, 2021). Three rabbits were exposed to 0.5 grams of barium chloride, moistened with water by application onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours after exposure. No skin irritation was caused by 4 hours exposure to barium chloride. Barium chloride was also non-irritant in the in vitro skin irritation study (Verbaan, 2010). The classification criteria according to regulation (EC) 1272/2008 as irritating to skin are not met, hence no classification is required for skin irritation.

 

Eye irritation:

Reference Stitzinger (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=3; effects were reversible within 21 days. The classification criteria according to regulation (EC) 1272/2008 as irritating to eyes are met since the mean score after 24, 48 and 72h was > 2 for chemosis and conjunctivae, hence classification and labelling as “skin irrit. 2” H319 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.