Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 December 2013 - 09 December 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Substance in 80% solution with propylene glycol in order to make the test feasable. The solvent is not thought to affect the results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

EC Number:

939-981-3

IUPAC Name:

Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

Constituent 2

Reference substance name:

Propane-1,2-diol

EC Number:

200-338-0

EC Name:

Propane-1,2-diol

Cas Number:

57-55-6

IUPAC Name:

propylene glycol

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch:
Not supplied
- Purity test date:
14th May 2013

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Test material prepared with 80% of substance and 20% propylene gylcol

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 13-EKIN-043
- Delivery date: 03 December 2013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca⁺⁺ and Mg⁺⁺ . Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to a second column of 3 wells containing 2 mL of maintenance medium in each well.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3mg/mL
- Incubation time: 3 hours at 37°C
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562nm
- Filter: No reference filter

NUMBER OF REPLICATE TISSUES: 3 for each testing group

For tjhe test item the relative mean tissue viabilities obtained after the 15-minute exposure period followed by the 42-hur post-exposure incubation period were compared to the mean of the negative control treated tissues.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed Tissues
- Procedure used to prepare the killed tissues (if applicable): Water-killed tissues were prepared by placing untreated EPISKIN™ tissues in a 12-well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37°C, 5% C02 in air for 48 ± 1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14°C to -30°C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature
- N. of replicates : 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be an irritant if relative mean tissue viability is ≤50%
- The test substance is considered to be a non-irritant to skin if relative mean tissue vialbility is >50%

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): 10µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10µL
- Concentration (if solution): 5% SDS

MTT: 0.3%

Duration of treatment / exposure:

Treatments: 22 minutes
MTT: 3 Hours

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative Control

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive Control

Value:

9.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not measured/tested

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item

Value:

24.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Direct reduction by the test item relative to the negative control value was 25.9% This is below the threshold value of 30% and is therefor acceptable
- Colour interference with MTT: MTT solution turned blue, indicating that the test item directly reduced MTT.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:

- Acceptance criteria met for negative control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was relative to the negative control treated tissues, and the standard deviation value of the percentage viability is
The mean OD562 for the negative control treated tissues was 1.165 and the standard deviation value of the percentage viability was 6.8%. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues was ≥0.6, and the standard deviation value of the percentage viability is ≤18%
The relative mean tissue viability for the positive control treated tissues was 9.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 0.6%. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for test item:
The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 11.3%. The test item acceptance criterion was therefore satisfied.
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item is classified as an irritant according to GHS criteria

Executive summary:

Introduction

The perecntage tissue viability was assessed for the test material using a reconstructed human epidermis model

Methods

The test was in line with the following guidelines:

• OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
• EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Conclusion

The test item is an irritant (Globally Harmonized Classification System - Category 2).