Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 25 September 2018 to 01 October 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Updated information. Study now available and provided ahead of extended submission date of 31/05/2019.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

EC Number:

271-672-2

EC Name:

Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

Cas Number:

68603-74-7

Molecular formula:

The substance is a UVCB so there is no molecular formula

IUPAC Name:

Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM™
- Source: SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): Lot no.: 18 EKIN 039 for live tissue, 18 EKIN 022 and 18 EKIN 029 for killed tissue.
- Date of initiation of testing: 25 September 2018

TEST SYSTEM CONDITIONS
- Incubation conditions prior to and post exposure: tissues were incubated in the dark at 37°C and 80 - 100% humidity in an atmosphere of 5 % CO2.
Incubation conditions for exposure: room temperature

TEST SYSTEM SET UP (PRE-INCUBATION)
Prior to the main test tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 23 hours at 37°C. Maintenance medium and Assay medium were supplied by the kit supplier, Skinethic Laboratories, Lyon, France.

APPLICATION OF TEST ITEM
No correction was made for the purity/composition of the test item. An excess amount of test item was applied by means of a filtration paper directly on top of the skin tissue and was added into 12-well plates. Three tissues were treated with 25 µL PBS (negative control) and 3 tissues with 25 µL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. The tissues were exposed to the test item, positive and negative controls for 15 ± 0.5 minutes.

In addition three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed.

POST-TREATMENT INCUBATION
Following rinsing the skin tissues were incubated for 42 hours at 37°C, 5% CO2, 80 - 100% humidity.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL MTT solution (0.3 mg/mL in phosphate buffered saline) per well
- Incubation time: 3 hours at 37°C
- MTT extraction solution: 500 µL isopropanol
- MTT extraction period: 69 hours refrigerated and protected from light
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: Three tissues per test item and postive and negative controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: Killed tissues
- N. of replicates : 3

PREDICTION MODEL / DECISION CRITERIA
As given in the test guideline (OECD 439)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item had a pasty consistence and an excess amount of the test item was applied directly on top of the skin tissue by means of a filtration paper.

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours at 37°C, 5% CO2, 80 - 100% humidity.

Number of replicates:

Three tissue replicates for the test itme and positive and negative controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three Replicates

Value:

35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The table below gives the mean tissue viability for the test item and postive and negative controls:

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative Control	100	5.7
Test Item	35	10
Positive Control	32	14

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item, Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates, was irritant in the in vitro skin irritation test under the experimental conditions used in the study. It meets the criteria for classification as a category 2 skin irritant according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

Introduction

The objective of this study was to evaluate the skin irritation potenetial of the test item, Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates for its ability to induce skin using the EPISKIN Small model™. The study was designed to meet the requirements of the following guidelines:

 

• OECD Guideline 439. In vitro Skin Irritation: Reconstructed Human Epidermis Test Method,(adopted 28 July 2015).
• EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In vitro Skin Irritation: Reconstructed Human Epidermis Model Test ". Official Journal of the European Union No. L142;Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

 

Method

 As the test item was a greasy solid so for application skin tissue was moistened with 5 µL of Milli-Q water and an excessive amount of the test item was applied directly on top of the skin tissue Testing was undertaken in triplicate. The skin tissue was exposed to the test item for 15 minutes after which the tissue was washed to remove residual test item. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed by measuring formazan production using an MTT assay. Positive and negative controls were also run.

The test item was found to interact with MTT. Therefore, in addition to the normal procedure, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was 21% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

Results

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 35%. Since the mean relative tissue viability for the test item was below 50% after 15 ± 0.5 minutes treatment it is considered to be an irritant.

Conclusion

The test item, Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates, was irritant in the in vitro skin irritation test under the experimental conditions used in the study. It meets the criteria for classification as a category 2 skin irritant according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.