Barium dibenzoate

Endpoint summary

Description of key information

Skin irritation: not irritating to the skin (OECD 439, GLP)

Eye irritation: causes serious eye irritation (OECD 405; GLP)

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:

2017-08-02 to 2017-08-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012-07-06

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2014-11-07

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry place at ambient temperature

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes

Cell source:

other: humans

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

other: Dulbecco's phosphate buffered saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT; MatTek)
- Tissue lot number: 25834

TEST FOR DIRECT MTT REDUCTION
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item was mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator (5 % CO2, 95 % RH).
- untreated MTT medium was used as control.
- if the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using freeze-killed tissues.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25 mg of the test item was mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min (5 % CO2, 95 % RH).
- if the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential using additional living tissues treated with the test item.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 ± 2 h.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature for 2 hours with shaking on a plate shaker.
Before using the extracts, the plate was shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. Optical density (OD) was measured with a filter band without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [mean ODtest item / positive control / ODmean of negative control] * 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues were calculated and used for classification according to the following prediction model:
Irritant potential of the test item will be predicted from the relative mean tissue viabilities compared to the negative control tissues. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (39 mg/cm²) of the test item

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL of the vehicle

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution

Duration of treatment / exposure:

60 ± 1 minute

Duration of post-treatment incubation (if applicable):

approx. 42 hours

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

95.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, no additional controls were necessary.
- Colour interference with MTT: mixture 25 mg of the test item per 300 µL aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, no additional controls were necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.773).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.4%)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.8% - 3.2%).
Please also refer to the field "An other information on results incl. tables" below.

Table 1: Result of the test item barium dibenzoate

Name	Negative Control (NK)			Positive Control (PC)			Test Material (TM)
Tissue	1	2	3	1	2	3	1	2	3
absolute OD570	1.755	1.853	1.950	0.112	0.104	0.087	1.640	1.762	1.755
	1.819	1.847	1.671	0.120	0.106	0.092	1.715	1.818	1.819
OD570(blank-corrected)	1.712	1.811	1.907	0.069	0.061	0.044	1.597	1.719	1.712
	1.776	1.804	1.628	0.078	0.063	0.049	1.672	1.775	1.776
mean OD570of the duplicates (blank-corrected)	1.744	1.808	1.767	0.073	0.062	0.047	1.635	1.747	1.744
total mean OD570of 3 replicate tissues (blank-corrected)	1.773*			0.061			1.692
SD OD570	0.032			0.013			0.056
relative tissue viability [%]	98.4	101.9	99.7	4.1	3.5	2.6	92.2	98.5	98.4
mean relative tissue viability [%]	100.0			3.4**			95.4
SD tissue viability [%]***	1.8			0.8			3.2
CV [% viabilities]	1.8			22.1			3.3

^* Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

^** Mean relative tissue viability of the three positive control tissues is ≤  20%.

^*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%

Table 2: Historical data

	Mean OD570±30nmNK	Mean Relative Viability [%] PC	SD Viability [%]
Mean	1.843	4.3	4.2
SD	0.286	2.2	4.7
n	22	22	84

Historical data were generatedfrom 2015 to 2017.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not irritating to the skin.

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:

2017-12-04 to 2017-12-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2017-10-09

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry place at ambient temperature

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 32 weeks old
- Weight at study initiation: 4.9 kg
- Housing: individually housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm²
- Diet (ad libitum): autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3 °C
- Relative humidity: 55 ± 10%
- Air changes: at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g of the test item in the conjunctival sac of one eye. Untreated eye served as control.

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

1, 24, 48 and 72 hours as well as 4 to 21 days after test item application

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

One female rabbit

Details on study design:

PREPARATION OF THE ANIMALS
Within 24 hours before the test and immediately prior to the application both eyes of each animal were examined.
Approx. 17 hours before the application the eyes were examined with the aid of a fluorescein solution (Fluoreszein SE Thilo®). The eyes were rinsed with physiological saline 0.9 % NaCl after the examination. None of the animals showed eye irritation, ocular defects, or pre-existing corneal injury.

INITIAL AND CONFIRMATORY TEST
The in vivo test was performed initially using one animal. The results of the initial test indicated that the test item is corrosive or severe irritant to the eye using the procedure described. Therefore, no additional animals were treated due to animal welfare reasons.

USE OF TOPICAL ANESTHETICS AND SYSTEMIC ANALGESICS
One hour before the application of the test item, 0.01 mg/kg of buprenorphine (Temgesic® 0.3 mg/mL) was administered subcutaneously in order to achieve a therapeutic level of systemic analgesia. Approx. 5 minutes prior to the application of the test item, 1-2 drops of an ocular anaesthetic (Proparakaine-POS® hydrochloride ophthalmic 0.5% solution) were administered in both the treated and the control eye of the animal.
To prevent pain and distress after the application of the test item the animal was treated with doses of buprenorphine and meloxicam (Metacam® 5 mg/mL) to provide a continued therapeutic level of systemic analgesia. Treatment with the analgesic medication was conducted from 11 hours post-application (day 0) upto 8 days post-application.

REMOVAL OF TEST SUBSTANCE
- Washing: treated eye was rinsed with physiological saline 0.9 % NaCl.
- Time after start of exposure: 1 hour after the application

SCORING SYSTEM: according to Draize scale

TOOL USED TO ASSESS SCORE: slit lamp biomicroscope / fluorescein
The treated eyes were examined with the aid of a fluorescein solution and a slit lamp biomicroscope 24 hours post-application and from then on daily until end of the observation period. The eyes were rinsed with physiological saline 0.9 % NaCl after the examination.

OBSERVATIONS
- body weight: prior to the administration and at least at the end of the observation period
- clinical observations: nature, severity and duration were recorded

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 13 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 11 days

Irritant / corrosive response data:

Animal no. 1:
- conjunctival redness: moderate conjunctival redness (grade 2) was observed from 1 hour until day 12 post-application. The effect was fully reversible within day 13 after application of the test material.

- conjuctival chemosis: moderate conjunctival chemosis (grade 3) was detected 1 hour post-application before severity level decreased from 24 until 48 hours post-application (grade 2) and from 72 hours until day 10 post-application (slight conjuncitvla chemosis; grade 1). The effect was fully reversible within day 11 after application of the test material.

- corneal opacity: slight corneal opacity (grade 1) was observed throughout the whole observation period of 21 days, except for day 14 and 15 after application. The effect was not reversible within 21 days.

- iris: the iris was not affected by administration of the test item.

Local effects were observed. The observed local effects mainly comprise severe to slight hypersecretion, red spot on the outer surface of the upper eyelid and vascularisation between eye rim and cornea lesion.
Upon fluorescein examinations starting 24 hours post-application, the treated eye of the animal showed corneal lesions (starting with approx. 40% of the area) which have not fully reversed within an observation period of 21 days.

Other effects:

- clinical observations: neither mortality nor significant clinical signs of toxicity were observed.
- body weight: the body weight development was not within the expected range.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The substance is serious eye damaging.
According to the EC Regulation No. 1272/2008 and subsequent adaptations, the substance is classified as a serious eye damaging (Category 1; H318).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Eye irritation

According to the bovine corneal opacity and permeability assay, since the IVIS was between 3 and 55, no prediction can be made regarding the test item barium dibenzoate.

Justification for classification or non-classification

Skin irritation

The substance does not possess a skin irritation potential based on an in vitro OECD 439 test and does not require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation

The substance does possess a serious eye damaging potential based on an in vivo OECD 405 test and does require classification as serious eye damaging according to Regulation (EC) No 1272/2008 and its subsequent adaptations (Category 1; H318).