[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 December 2015 - 04 December 2015 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (28 July 2015)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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 (REACH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine

Test material form:

solid: pellets

Details on test material:

- Storage condition of test material: Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

other: EpiDerm™ Reconstructed Human Epidermis Model Kit

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SITE
- Area of exposure: 0.63cm²
- % coverage: 100%

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item.
- Time after start of exposure: 3 or 30 min

SCORING SYSTEM: Quantitative MTT Assessment (percentage tissue viability)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg, ground to a fine powder before use

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

Duration of treatment / exposure:

3 min or 60 min

Duration of post-treatment incubation (if applicable):

3-Hour MTT incubation

Number of replicates:

each two wells for the test item, positive and negative control, for both 3 min and 60 min exposure, resulting in a total of 12 wells

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: cell viability, percentage of control

Run / experiment:

mean after 3 min

Value:

110.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

6.9 after 30 min (cell viability, percentage of control)

Results 2

Irritation / corrosion parameter:

other: cell viability, percentage of control

Run / experiment:

mean after 60 min

Value:

103.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

5.6 after 60 min (cell viability, percentage of control)

Other effects / acceptance of results:

The test item was considered to be non-corrosive to the skin.

Any other information on results incl. tables

RESULTS

 

Direct MTT Reduction

An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze killed tissues for quantitative correction of results or for reporting purposes.

 

Assessment of Color Interference with the MTT endpoint

The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

 

Test Item, Positive Control Item and Negative Control Item

Mean OD₅₆₂ values and viabilities for the negative control, positive control and test item are given in the table.

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	6.9	110.4
60 minute	100*	5.6	103.7

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

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

Tissue	Exposure Period	Mean OD562 of individual tissues	Mean OD562 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 min	1.836	1.722	0.161	9.4	100*
		1.608
	60 min	1.780	1.830	0.070	3.8
		1.879
Positive Control	3 min	0.116	0.119	0.004	N/A	6.9
		0.121
	60 min	0.102	0.102	0.000	N/A	5.6
		0.102
Test Item	3 min	1.882	1.902	0.028	1.5	110.4
		1.921
	60 min	1.763	1.897	0.189	10.0	103.7
		2.030

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

 

Relative mean % tissue viability = (mean OD₅₆₂ of test item / mean OD₅₆₂ of negative control) * 100

 

Coefficient of Variation = (standard deviation / mean OD₅₆₂ of duplicate tissues) * 100

Quality Criteria

The mean OD₅₆₂ for the negative control treated tissues was 1.722 for the 3 Minute exposure period and 1.830 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.

 

The relative mean tissue viability for the positive control treated tissues was 5.6% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.

 

In the range 20-100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

 

CONCLUSION

The test item was considered to be non-corrosive to the skin.

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Conclusions:

The study was conducted under GLP according to OECD guideline 431 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the corrosive potential of Octadecanoic acid, reaction products with tetraethylenepentamine to the skin in vitro. The viability of the cells treated with the test item was ≥ 50% (3 min treatment) and ≥ 15% (60 min) of control. Hence, the test item was considered to be non-corrosive to the skin.
In a tiered in vitro testing strategy, this result (non-corrosive) may however only give an indication of the skin-damaging potential, but does not allow a definitive classification according to Regulation 1272/2008. So, an additional skin irritation test according to OECD 439 should be conducted.

Executive summary:

The purpose of this OECD TG 431 GLP study is to evaluate the corrosivity potential of Octadecanoic acid, reaction products with tetraethylenepentamine 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.

 

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 directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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 µL 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).

 

The relative mean viabilities for each treatment group were110.4% (3 min) and 103.7% (60 min) of control.

 

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

 

The test item was considered to be non-corrosive to the skin.