Amines, rosin

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-25 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

GLP compliance:

yes

Remarks:

Except for the following: - Information about purity/composition of the test item was not available prior to completion of the study. - The quality environment in which the characterisation of the test item was performed was not known.

Test material

Test material information

Constituent 1

Reference substance name:

Amines, rosin

EC Number:

263-139-8

EC Name:

Amines, rosin

Cas Number:

61790-47-4

Molecular formula:

C19H31N1

IUPAC Name:

1-[(1R,4aR,10aS)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl]methanamine; 1-[1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl]methanamine

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rosin, Batch: 542700
- Appearance: Transparent pale amber viscous liquid
- Expiration date of the lot/batch: 31 March 2016
- Purity test date: Not indicated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: EPI-200, normal, human-derived epidermal keratinocytes

Cell source:

other: MatTek Corporation, Ashland MA, U.S.A.

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Recommended test system in international guidelines (OECD and EC)

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION:
EpiDerm Skin Model (EPI-200, Lot no.: 22676 kit K and kit J). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to
form a multilayered, 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 EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Environmental conditions:
- Prior to the assay, skin tissues were kept refrigerated from the day they were received.
- All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 58 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C
(actual range 36.3 - 37.3°C).
- Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. - Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door.
- Based on laboratory historical data these deviations are considered not to affect the study integrity.

APPLICATION OF TEST SUBSTANCE
- 1 hour before the start of the assay, tissues were transferred to 6-well plates containing 0.9 mL DMEM medium per well. The plates were incubated for approximately 1.5 hours at 37 ± 1°C
- The medium was replaced with fresh DMEM medium just before Rosin Amine 90 was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control.
- Two tissues were used for a 3-minute exposure to Rosin Amine 90 and two for a 1-hour exposure. Fifty µl of the undiluted test item was added into the 6-well plates on top of the skin tissues.
- The remaining tissues were treated with 50 µl Milli-Q water (negative control) and with 50 µl 8N KOH (positive control), respectively.
- Following the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 µl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.
 
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporatio.

CELL VIABILITY MEASURMENT / NUMBER OF INDEPENDENT TESTING RUNS
The DMEM medium was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
 
Test for the interference of the test item with the MTT endpoint:
A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

PREDICTION MODEL / DECISION CRITERIA:
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430:
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- 50 µl of the undiluted test item was added into the 6-well plates on top of the skin tissues

NEGATIVE CONTROL
- 50 µl Milli-Q water

POSITIVE CONTROL
- th 50 µl 8N KOH

After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. Rinsed tissues were kept in 24 well plates on
300 µl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

Duration of treatment / exposure:

3-minute and 1-hour exposure

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 72

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

15%

Remarks on result:

other: not corrosive

Remarks:

3-minute application viability (percentage of control)

Results 2

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 48

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

11%

Remarks on result:

other:

Remarks:

1-hr application viability (percentage of control)

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Rosin Amine 90 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Rosin Amine 90 did not interfere with the MTT endpoint.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 15%. The maximum inter-tissue variability in viability between two tissues treated identically was less than 23% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 13%. It was therefore concluded that the test system functioned properly.

ACCEPTANCE OF RESULTS:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 3-minute exposure to the positive control should be ≤ 30%.
c) In the range of 20 – 100% viability, the maximum inter-tissue variability (in viability) is ≤ 30% between two tissues treated identically.
d) In the range of 20 – 100% viability, the maximum difference in percentage between the mean
viability of two tissues and one of the two tissues is ≤ 15%.

Applicant's summary and conclusion

Interpretation of results:

other: Rosin Amine 90 is considered to be not corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

It is concluded that this test is valid and that Rosin Amine 90 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.

Executive summary:

The objective of this study was to evaluate the ability of the test substance, Rosin Amine 90, to induce skin corrosion when applied topically on a human three dimensional epidermal model. The study was conducted in accordance with OECG Guideline 431 and EC Commission Regulation No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: 'In vitro Skin Corrosion: Human Skin Model Test'.

The possible corrosive potential of Rosin Amine 90 was tested through topical application for 3 minutes and 1 hour. The test substance was applied undiluted (50 µL) on top of the skin tissue and then following the exposure period, the tissues were washed with phosphate buffered saline. Rinsed tissues were kept in 24 -well plates on 300 µl DMEM medium until 6 tissues were dosed and rinsed (6 tissues = 1 application time).

Rosin Amine 90 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Since the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Rosin Amine 90 did not interfere with the MTT endpoint.

The positive control had a mean relative tissue viability of 15% after 3 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The acceptability criteria for the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues were met, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3 -minute and 1 -hour treatments with Rosin Amine 90 compared to the negative control tissues was 72% and 48% respectively. Because the mean relative tissue viability for Rosin Amine 90 was not below 50% after the 3 -minute treatment and not below 15% after the 1 -hour treatment Rosin Amine 90 is considered to be not corrosive.

Finally, it is concluded that this test is valid since the acceptability criteria were met and that Rosin Amine 90 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.