1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives (upper limit: 41% w/w; typical concentration: 38% w/w)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental stat date: 04/11/14 Experimental completion date: 06/11/14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

other: LIQUID

Details on test material:

Identification: PP-R-001 (also known as Trigonox 501-CS40)
Chemical name: 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris (Ethyl and Propyl) derivatives
IUPAC Name: Reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl- 1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3, 6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane
CAS Number: 1613243-54-1
Batch Number: BYK004856-98
Purity: not applicable
Physical state / Appearance: clear, colorless liquid
Expiry Date: 03 April 2017
Storage Conditions: approximately 4 °C in the dark

Test animals

Species:

other: EPIDERM (TM) Reconstructed Human Epidermis Model Kit

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

Supplier : MatTek Corporation, Ashland, MA, USA
Date received : 04 November 2014
EpiDermTM Tissues (0.5cm2) lot number it K
Assay Medium lot number : 103014ZSD

Upon receipt of the EpidermTM tissues, the sealed 24-well plate was placed into a refrigerator overnight.

Test system

Type of coverage:

other: Test item applied to culture surface, at air interface

Preparation of test site:

other: The assay medium was pre-warmed before use. 0.9 mL of assay medium was pipetted into the appropriate wells of 2 pre-labeled 6-well plates for both the 3 Min, and 60 Min, exposure periods. EpiDerm tissues were transferred into the 6 well plate

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative Control: Sterile Distilled Water Positive Control: 8.0N Potassium Hydroxide

Amount / concentration applied:

Amount applied: 50 µL per well

Concentration: not stated in study report4

Duration of treatment / exposure:

3 minutes and 60 minutes

Observation period:

Immediate observation following exposuremn

Number of animals:

Two x 24-well plates

Details on study design:

Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Dye Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.

One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:

100 L of the test item was added to 900 L of a 1.0 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 C, 5% CO2 in air for 3 hours. 100 L of sterile distilled water was tested concurrently to act as a control.

If the MTT solution containing the test item turns blue relative to the control, the test item was presumed to have reduced the MTT.


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 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 were placed into a refrigerator overnight, 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 blue formazan extract was 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 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

79.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: reversibility not determined

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

87.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: reversibility not determined

Other effects / acceptance of results:

The relative mean viability of the test item treated tissues was as follows:

60 minutes exposure : 79.3%
3 minutes exposure : 87.1%

Direct MTT Reduction
The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.

Any other information on results incl. tables

TABLE

Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	Exposure Time	Mean OD5621	% Viability
Negative Control	3 minute	2.389	100*
	60 minute	2.417	100*
Positive Control	3 minute	0.123	5.1
	60 minute	0.054	2.2
Test Item	3 minute	2.080	87.1
	60 minute	1.917	79.3

 

 

*=   The mean viability of the negative control tissues is set at 100%

1=   Mean of EpiDerm tissues tested in duplicate

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 5.1 % relative to the negative control treated tissues following the 3‑Minute exposure period. The positive control acceptance criterion was therefore satisfied.

 

The mean OD₅₆₂for the negative control treated tissues was 2.389 for the 3‑Minute exposure period and 2.417 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

The test item, PP-R-001 (CAS#1613243-54-1), was considered to be non-corrosive to the skin.

Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item, PP-R-001 (CAS#1613243-54-1), using the EPIDERM™ Human Skin Model after treatment periods of 3 and 60 minutes.

 

Corrosion is directly related to cytotoxicity in the EpiDerm tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

 

 Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. 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 562 nm (OD₅₆₂).

 

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

  

Results

The relative mean viabilities of the test item treated tissues were:

 

60 minutes exposure		:		79.3%
3 minutes exposure		:		87.1%

 

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

  

Conclusion

The test item, PP-R-001 (CAS#1613243-54-1), was considered to be non-corrosive to the skin.