Resin acids and Rosin acids, barium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb - Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Resin acids and Rosin acids, barium salts

EC Number:

269-142-0

EC Name:

Resin acids and Rosin acids, barium salts

Cas Number:

68188-14-7

Molecular formula:

N/A

IUPAC Name:

Resin acids and Rosin acids, barium salts

Test material form:

solid

Details on test material:

Specific details on test material used for the study
SOURCE OF TEST MATERIAL
- Batch number of test material: EXP NR.3757-01
- Expiration date of the lot/batch: 31 Dec 2029
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator
- Stability under storage conditions: The stability of the test substance under storage conditions is
guaranteed until 31 Dec 2029 as indicated by the sponsor, and the sponsor holds this responsibility.
- Stability under test conditions: no data

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen Germany
- lot/batch number of test material: EXP NR.3757-01
- Expiration date: 31 Dec 2029
- Purity: 100 % UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator
- Stability under storage conditions: The stability was guaranteed over the study period

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi 200
- Tissue batch number(s): 30854

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C, 1 hour
- Post-incubation temperature and time: 37°C, 18 ± 2 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: yes
- 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: 1.0 mg / mL MTT diluent
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD570, accetable
- Barrier function: Lower acceptance limit: ET50 = 4.0 hours, Upper acceptance limit: ET50 = 8.7 hours
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- freeze-killed tissues
- Procedure used to prepare the killed tissues: freezing
- N. of replicates : 3
- Method of calculation used: The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of three tissues treated in the same way is calculated.

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 irritant to skin if the mean tissue viability after exposure is less than 50%.
- The test substance is considered to be borderline irritant (inconclusive) to skin if the mean tissue viability after exposure is 45-55%.
- The test substance is considered to be non-irritant to skin if the viability after exposure is greater than or equal to 50%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: The “borderline“ evaluation (50 ± 5%) was statistically determined by using historic BASF data and hence considers the variance of the test method. This evaluation is confirming the borderline range provided in OECD Guideline 439.

Control samples:

yes, concurrent negative control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg per tissue
- Concentration (if solution):

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

25 minutes at room temperature and 35 minutes in the incubator

Duration of post-treatment incubation (if applicable):

ca. 42

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1

Value:

104.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2

Value:

99.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3

Value:

96.1

Vehicle controls validity:

not applicable

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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

Table 1: Individual and mean OD570 values, individual and mean viability values, standard deviations

and coefficient of variation

Test substance identification			tissue 1	tissue 2	tissue 3	mean	SD	CV [%]
NC	viable tissues	mean OD570	2.118	2.093	2.054	2.088
		viability [% of NC]	101.4	100.2	98.3	100.0	1.5	1.5
	KC tissues	mean OD570	0.059	0.072	0.054	0.061
		viability [% of NC]	2.8	3.4	2.6	2.9	0.4	15.2
Barium resinate	viable tissues	mean OD570	2.189	2.085	2.008	2.094
		viability [% of NC]	104.8	99.8	96.1	100.3	4.4	4.3
	KC tissues*	mean OD570KC NC corrected	0.000	0.000	0.000	0.000
		viability [% of NC]	0.0	0.0	0.0	0.0	0.0
	Final mean viability of tissues after KC correction [% of NC]:					100.3
PC	viable tissues	mean OD570	0.043	0.043	0.049	0.045
		viability [% of NC]	2.1	2.0	2.3	2.1	0.2	8.1

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed and by applying the evaluation criteria described, it was concluded that the test substance does not show a skin irritation potential in the EpiDermTM in vitro skin irritation test under the test conditions chosen.

Executive summary:

The potential of the test substance to cause dermal irritation was assessed by a topical application of ca. 2 x 25 µL bulk volume of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). Due to the chemical and physical properties of the test substance, a double bulk volume (corresponding to ca. 10 mg per tissue) was required for covering the whole tissue surface.

Three EpiDerm™ tissues were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. In addition to the test substance, 30 µL per tissue of a negative control (PBS) and of a positive control (5% SDS) were applied to three tissues each.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of both values indicates the relative tissue viability.  

The following results were obtained in the EpiDerm skin irritation test:

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced. The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 100.3%. The acceptance criteria for the variability of the tissues are met. Application of the positive control 5% SDS showed a relative mean viability of the tissues of 2.1% and reflects the expected sensitivity of the tissues. The mean OD570 of the negative control (PBS) fulfills the acceptance criteria and demonstrates the validity of the assay.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation test under the test conditions chosen.