Resin acids and Rosin acids, maleated, sodium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-06-11 to 2014-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in Section 13.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2014-06-11 to 2014-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in Section 13.

Reason / purpose for cross-reference:

read-across source

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

107.26

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Cell viability after 3 mins was 107.26%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

173.42

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Cell viability after 60 mins was 173.42%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: The colour in both wells treated with the test substance was Yellow, meaning that the test substance does not interact with MTT
- Colour interference with MTT: At the end of the shaking period, the solution did not change colour, meaning that no additional inserts were required to perform the skin corrosion assay.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, Observed OD570 value was 1.79 (3 mins) and 1.58 (60 mins). Acceptance criteria: ≥ 0.8 and ≤ 3
- Acceptance criteria met for positive control: Yes, Observed value for mean viability was 0.63. Acceptance criteria: < 15% after 1 hour

Table 1. Acceptance Criteria and Results
Acceptance Criteria	Observed Values	Result
Mean OD570of Negative Control ≥ 0.8 and ≤ 3	1.79 (3 mins) 1.58 (60 mins)	Pass
Mean Viability of Positive Control compared to Negative Control < 15% after 1 hour	0.63	Pass
Test Substance acceptability criteria: Standard deviation of Mean Value expressed in % should be ≤ 30%.	21.21 (3 mins) 4.95 (60 mins)	Pass
The mean of blanks (Mean Blank) will be calculated. The OD of the mean blank should not exceed 0.1.	0.04 (3 mins) 0.04 (60 mins)	Pass
The OD of the Negative Control should be 20 fold greater than the OD of the blank.	44.75 (3 mins) 39.50 (60 mins)	Pass

Interpretation of results:

GHS criteria not met

Remarks:

Non Corrosive under EU CLP

Conclusions:

On the basis of the results obtained in the skin corrosion assay, the test substance was considered to be non corrosive.

Executive summary:

This data is being read across from the source study that tested Resin acids and Rosin acids, maleated, potassium salts based on category read across that is explained in the category justification document attached in Section 13 of the dossier.

In a GLP-compliant, key Guideline (OECD 431) In Vitro skin corrosion study, the test material (Resin acids and Rosin acids, maleated, potassium salts (CAS# 85409-27-4) was applied to the stratum corneum of an epidermal model (SkinEthics^TM RhE; 2 epidermis units per test substance) for 2 different exposure periods: 3 minutes and 60 minutes. Exposure to the test material was terminated by rinsing with D-PBS with Ca⁺²/ Mg⁺². KOH 8N was used as the positive control and sterile ddH₂O was used as the negative control.

 

The viability of the epidermis was assessed by measuring the mitochondrial activity by MTT assay. The treated issues were incubated for 3 hours ± 15 minutes with a 1 mg/mL MTT solution at 37°C, 5% CO₂, and 95% RH, 0.3 mL per well. The precipitated formazan salts were extracted overnight at 4°C by using isopropanol (1.5 mL/well) and quantification performed spectrophotometrically at 570 ± 30 nm.

 

The test material did not interact with MTT and with the mesh and there was no colour interference observed. The test material, negative control, and positive control met the acceptance criteria for this assay. Cell viability (expressed in terms of percentage compared to the viability of the negative control (set as 100%)) was observed to be 107.26 % after 3 minute exposure and 173.42 % after 60 minute exposure to the test material.

 

On the basis of the results obtained in the skin corrosion assay, Resin acids and Rosin acids, maleated, potassium salts was considered to be non-corrosive.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Resin acids and Rosin acids, maleated, potassium salts

EC Number:

287-094-9

EC Name:

Resin acids and Rosin acids, maleated, potassium salts

Cas Number:

85409-27-4

Molecular formula:

not applicable as the substance is a UVCB

IUPAC Name:

Resin acids and Rosin acids, maleated, potassium salts

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Mare Spa (Italy); Batch no. 229-080513
- Expiration date of the lot/batch: 2014-07-23
- Purity test date: 2014-04-24

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

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Amber liquid

OTHER SPECIFICS:
pH (8%): 12.5

In vitro test system

Test system:

human skin model

Remarks:

SKINETHIC RhE: Reconstructed Human Epidermis

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Origin foreskin (2 years), Reconstructed Human Epidermis

Details on animal used as source of test system:

SOURCE
- Source: Inserts SkinEthic RhE supplied by SkinEthic

Justification for test system used:

The RhE model presents a histological morphology comparable to the in vivo human tissue.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RhE
- Tissue batch number(s): 14 RHE 0511
- Production date: 2014-06-16
- Shipping date: 2014-06-01
- Delivery date: ???
- Date of initiation of testing: 2014-06-17

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 washings with 1 mL of Dulbecco's Phosphate Buffered Saline (D-PBS) with Ca+2/Mg+2 (ID 2919), 5-8 cm distance from the insert.
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Microplate reader SRA 37
- Wavelength: 570 ± 30 nm
- Filter: without reference filter
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: 540-600 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Acceptance criteria: OD Values between 0.6 and 1.5.
- Barrier function: Acceptance criteria: 4 hours - 10 hours upon treatment with 1% Triton X-100.
- Morphology: Acceptance Criteria: evidence of multi-layered human epidermis-like structure (at least 4 viable cell layers)
- Contamination: Inserts tested for the absence of HIV-1 and HIV-2, hepatitis C and B and syphillis. Inserts certified bacteria and mycoplasma free.

NUMBER OF REPLICATE TISSUES: 2 per exposure time

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Test material did not interfere with MTT and with the mesh hence 12 inserts were ordered to carry out the test.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Not specified

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: Not applicable

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 40 µL of the test material were applied to each insert.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL of Sterile ddH2O

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL of 8 N KOH solution

Duration of treatment / exposure:

1 hour (exposure time 1) or 3 minutes (exposure time 2)

Duration of post-treatment incubation (if applicable):

For MTT Cell Viability evaluation, plates were incubated for 3 hours at 37°C ± 1°C, 5% CO2, 95% RH.

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

107.26

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Cell viability after 3 mins was 107.26%

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

173.42

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Cell viability after 60 mins was 173.42%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: The colour in both wells treated with the test substance was Yellow, meaning that the test substance does not interact with MTT
- Colour interference with MTT: At the end of the shaking period, the solution did not change colour, meaning that no additional inserts were required to perform the skin corrosion assay.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, Observed OD570 value was 1.79 (3 mins) and 1.58 (60 mins). Acceptance criteria: ≥ 0.8 and ≤ 3
- Acceptance criteria met for positive control: Yes, Observed value for mean viability was 0.63. Acceptance criteria: < 15% after 1 hour

Any other information on results incl. tables

Table 1. Acceptance Criteria and Results
Acceptance Criteria	Observed Values	Result
Mean OD570of Negative Control ≥ 0.8 and ≤ 3	1.79 (3 mins) 1.58 (60 mins)	Pass
Mean Viability of Positive Control compared to Negative Control < 15% after 1 hour	0.63	Pass
Test Substance acceptability criteria: Standard deviation of Mean Value expressed in % should be ≤ 30%.	21.21 (3 mins) 4.95 (60 mins)	Pass
The mean of blanks (Mean Blank) will be calculated. The OD of the mean blank should not exceed 0.1.	0.04 (3 mins) 0.04 (60 mins)	Pass
The OD of the Negative Control should be 20 fold greater than the OD of the blank.	44.75 (3 mins) 39.50 (60 mins)	Pass

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Non Corrosive under EU CLP

Conclusions:

On the basis of the results obtained in the skin corrosion assay, the test substance was considered to be non corrosive.

Executive summary:

In a GLP-compliant, key Guideline (OECD 431) In Vitro skin corrosion study, the test material (Resin acids and Rosin acids, maleated, potassium salts (CAS# 85409-27-4) was applied to the stratum corneum of an epidermal model (SkinEthics^TM RhE; 2 epidermis units per test substance) for 2 different exposure periods: 3 minutes and 60 minutes. Exposure to the test material was terminated by rinsing with D-PBS with Ca⁺²/ Mg⁺². KOH 8N was used as the positive control and sterile ddH₂O was used as the negative control.

 

The viability of the epidermis was assessed by measuring the mitochondrial activity by MTT assay. The treated issues were incubated for 3 hours ± 15 minutes with a 1 mg/mL MTT solution at 37°C, 5% CO₂, and 95% RH, 0.3 mL per well. The precipitated formazan salts were extracted overnight at 4°C by using isopropanol (1.5 mL/well) and quantification performed spectrophotometrically at 570 ± 30 nm.

 

The test material did not interact with MTT and with the mesh and there was no colour interference observed. The test material, negative control, and positive control met the acceptance criteria for this assay. Cell viability (expressed in terms of percentage compared to the viability of the negative control (set as 100%)) was observed to be 107.26 % after 3 minute exposure and 173.42 % after 60 minute exposure to the test material.

 

On the basis of the results obtained in the skin corrosion assay, Resin acids and Rosin acids, maleated, potassium salts was considered to be non-corrosive.