Barium(2+) 12-hydroxyoctadecanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-10-11 to 2017-10-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 April 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Barium(2+) 12-hydroxyoctadecanoate

EC Number:

244-469-1

EC Name:

Barium(2+) 12-hydroxyoctadecanoate

Cas Number:

21598-22-1

Molecular formula:

C18H36O3.1/2Ba

IUPAC Name:

barium bis(12-hydroxyoctadecanoate)

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: Lab NP_20171034-003
- Expiration date of the lot/batch: 2022-08-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: closed vessel at room temperature (20±5°C).

OTHER SPECIFICS:
white solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin SM
- Tissue batch number: 17-EKIN-041

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time, the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis).
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT
- Incubation time: 3 h
- Spectrophotometer: 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC)
- Wavelength: 570 ± 10 nm
- Linear OD range of spectrophotometer: Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752)

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 15 minutes exposure is equal or less than 50%.
- The test substance is considered to be non-corrosive to skin if the viability after 15 minutes exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

104

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Validity of the test

The mean OD value of the three negative control tissues was 0.897. The mean OD value obtained for the positive control was 0.166 and this result corresponds to 19 % viability when compared to the results obtained from the negative controls. Each calculated standard deviation value (SD) for the % viability was below 18.All validity criteria were within acceptable limits and therefore the study can be considered as valid.

 

Possible direct MTT reduction with test substance

No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be excluded.

Colouring potential of test substances

The test item showed no ability to become coloured in contact with water. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Table 1: Summary of the results

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	0.874	97
	2	0.853	95
	3	0.965	108
	mean	0.897	100
	standard deviation (SD)		6.62
Positive Control: SDS (5 % aq.)	1	0.136	15
	2	0.181	20
	3	0.181	20
	mean	0.166	19
	standard deviation (SD)		2.95
Test Item:	1	0.971	108
	2	0.892	99
	3	0.931	104
	mean	0.931	104
	standard deviation (SD)		4.40

Table 2: Historical Control Data (Period of 2011-2017 October)

	Negative Control data	Positive Control data
	Phosphate Buffered Saline (1 x PBS)	Sodium Dodecyl Sulphate (SDS) 5 % aq. solution
	Optical Density (OD)	Optical Density (OD)	Viability (% control)
Mean	0.828	0.111	14
Minimum	0.555	0.015	2
Maximum	1.414	0.299	39

Table 3: Quality control of the Episkin SM

Test	Specification	Result
Histology scoring (HES stained vertical paraffin sections)	≥ 19.5	23.4 ± 0.4
IC50 determination (SDS concentration, MTT test)	Well differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.	Statisfactory
	1.5 mg/mL ≤ IC50 ≤ 3.0 mg/mL	1.9 mg/mL

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In the in vitro skin irritation assay (Episkin) according to OECD guideline 439, a mean tissue viability of 104 % was determined.

Executive summary:

The skin irritating potential of the test item was assessed in an in vitro skin irritation assay (Episkin) according to OECD guideline 439. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. 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 at 37 °C in 5 % CO2 and protected from light. The resulting formazan crystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 104 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.