Tall oil pitch, reaction product with triethylene glycol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2017 - 03 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Deviations:

yes

Remarks:

Deviations were considered to have not affected the integrity or validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008

Version / remarks:

Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

yes

Remarks:

Deviations were considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Not available

Cas Number:

2135769-54-7

Molecular formula:

NA

IUPAC Name:

Not available

Test material form:

liquid: viscous

Details on test material:

Vapour pressure: 3.9 x 10-3 Pa
Water solubility: TBC
Density: 1.0
Appearance: black viscous liquid
Melting point: -52 to -21 °C
Boiling point: no boiling and no decomposition up to 300°C

Specific details on test material used for the study:

Identification: Tall Oil Pitch, reaction product with triethylene glycol
Batch: LABO 16-02
Purity: 100%
Physical state/Appearance: brown viscous liquid
Expiry Date: 14 November 2018
Storage Conditions: room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

other: Reconstructed Human Epidermis Model Kit

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek

Source strain:

not specified

Justification for test system used:

This test is able to reliably discriminate chemicals that are corrosive to skin from non-corrosive chemicals, and can therefore be used for the classification of skin corrosion hazard according to the GHS System adopted by the OECD

Vehicle:

not specified

Details on test system:

Pre-Incubation
The assay medium was pre warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 Minute and 60 Minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing
Before pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 Minute and 60 Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24 well plate was prepared for the MTT loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 Minute exposure period was returned to the incubator, while the other was being dosed for the 60 Minute exposure. For the 60 Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period.
When dosing for the 60 Minute exposure period was complete, the same procedure was repeated for the 3 Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24 well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3 Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

Absorbance/Optical Density Measurements
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount and concentration (s) applied (volume or weight with unit): 50 µL of the test item was applied to the corresponding tissues

POSITIVE CONTROL
- Amount(s) applied (volume or weight) and concentrations: 8.0 N Potassium Hydroxide (positive control) were applied to the corresponding tissues
Batch: SLBM9898V
Purity: 7.92M
Expiry Date: 7 April 2020
Storage Conditions: room temperature
Supplier: Sigma-Aldrich

NEGATIVE CONTROL
- Amount(s) applied (volume or weight) and concentrations: 50 µL
Identification: Sterile distilled water
Batch:3012436
Purity: not applicable
Expiry Date: 01 October 2018
Storage Conditions: room temperature
Supplier: Aguettant Ltd

Duration of treatment / exposure:

3 Hour incubation

Number of replicates:

two tissue replicates of each treatment group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

99.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

100.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Tall Oil Pitch, reaction product with triethylene glycol was considered to be non-corrosive to the skin, according to the result of this study.

Executive summary:

The aim of this study was to determin the corrosivity potential of the test item Tall Oil Pitch, reaction product with triethylene glycol using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. The study was conducted according to the OECD 471 Guideline and under GLP conditions.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to have the potential to cause color interference with the MTT end-point therefore additional tissues were incorporated into the testing to correct for this. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD₅₇₀).

The quality criteria required for acceptance of results in the test were satisfied.

The results of the study showed that the test itemTall Oil Pitch, reaction product with triethylene glycol is not corrosive.