Resin acids and Rosin acids, reaction products with formaldehyde, calcium 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:

From 25 Sep 2012 to 18 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

July 22, 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: B.46: In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test; Official Journal of the European Union, No. L 193

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Resin acids and Rosin acids, reaction products with formaldehyde, calcium salts

EC Number:

696-130-6

Cas Number:

1364681-45-7

Molecular formula:

Unspecified

IUPAC Name:

Resin acids and Rosin acids, reaction products with formaldehyde, calcium salts

Test material form:

solid - liquid: suspension

Details on test material:

- Name of test material (as cited in study report): Resin acids and rosin acids, reaction products with formaldehyde, calcium salts
- Physical state: Solid
- Analytical purity: 96.0 % w/w
- Lot/batch No.: DMG 7245
- Expiration date of the lot/batch: July 05, 2016
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature
- Homogeneity: The test substance was homogeneous by visual inspection.
- Others:
pH-value: Ca. 5 (undiluted test substance, moistened with water)

Test animals

Species:

other: in vitro test on three dimensional human epidermis model (EpiDerm™ model which consists of normal human-derived epidermal keratinocytes cultured to form a multi layered, highly differentiated model of the human epidermis.)

Strain:

other: not applicable

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable (in vitro test)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL bulk volume (about 5 mg)

Duration of treatment / exposure:

1 hour followed by a 42-hours post-incubation period

Observation period:

not applicable (in vitro test)

Number of animals:

not applicable (in vitro test)

Details on study design:

Test system:
- In vitro test system on three dimensional human epidermis models. The EpiDermTM model consists of normal, human-derived epidermal keratinocytes cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
- The test system is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed epidermis after a 1-hour topical exposure and about 42 hours postincubation.

Material and technical equipment:
- EpiDerm™ 200 kit: MatTek Corp., Bratislava, Slovakia containing 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® diameter 1 cm.

Controls:
- Negative control (NC): 30 μL PBS, sterile
- Positive control (PC): 30 µL 5% (w/v) sodium dodecyl sulfate (SDS, Sigma, Germany) in deionized water, sterile

Experimental procedure:
- Direct MTT reduction:
To assess the ability of the test material to directly reduce MTT a pretest was performed. Thereby, the test substance was added to 0.9 mL of the MTT solution. A negative control (de-ionized water) was tested concurrently. If the MTT solution colour or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the colour density produced by metabolic capacity of the tissue and would falsify the test results.
- Basic procedure:
Three tissues were treated with the test substance, the PC and NC, respectively.
• 25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid.
• 1 hour after start of application of the test material to the stratum corneum surface of the EpiDermTM tissue, residual test material was removed with sterile PBS and the surface of each tissue was dried. Subsequently, the tissues were incubated at 37°C for 24 ± 2 hours, transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and thereafter placed into the incubator for additional 18 ± 2 hours post-incubation period.
• After the postincubation period induced tissue destruction (cytotoxicity = loss of viability) was determined by measuring the metabolic activity of the tissue using a colourimetric assay. Thereby, cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow coloured water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue coloured formazan. After isopropanol extraction of the formazan from the tissues, the optical densitiy of the extract is determined spectrophotometrically. Optical density of the extracts of test substance treated tissues is compared to negative control values and expressed as relative tissue viability.

Acceptance criteria:
In case one of the below given acceptance criteria is not covered, repetition of the test is considered.
- Assay acceptance criterion for the negative control (NC): The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.
- Assay acceptance criterion for the positive control (PC): 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.
- Assay acceptance criterion for tissue variability: For every treatment, 3 tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of %-viability is ≤ 20.

Evaluation criteria:
- Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: viability (%)

Value:

102

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 h. Max. score: 102.7. Reversibility: other: not applicable. Remarks: In Vitro Skin Irritation. (migrated information)

In vivo

Irritant / corrosive response data:

The test substance is not able to reduce MTT directly.
- The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 102%.

Other effects:

No other effects observed.

Any other information on results incl. tables

Table 2: In Vitro Skin Irritation

Test substance		Exposure: 1 hour
		Tissue 1	Tissue 2	Tissue 3	Mean	SD
NC	Mean OD570	2.295	2.144	2.121	2.187
	Viability (% of NC)	105.0	98.1	97.0	100	4.33
Test Substance	Mean OD570	2.222	2.237	2.245	2.234
	Viability (% of NC)	101.6	102.3	102.7	102	0.54
PC	Mean OD570	0.059	0.063	0.065	0.062
	Viability (% of NC)	2.7	2.9	2.9	3	0.13

NC: Negative control, PBS, sterile 

PC: Positive control, 5% SDS in deionized water, sterile

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information

Conclusions:

CLP: not classified
DSD: not classified