Reaction products of propane-1,2-diamine with 2-[(2-methylphenoxy)methyl]oxirane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-10-07 to 2016-02-08

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

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiSkin SOP, Version 1.8 (February 2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-((2-Amino-2-methylethyl)amino)-3-(2-methylphenoxy)propan-2-ol

Molecular formula:

C13H22N2O2

IUPAC Name:

1-((2-Amino-2-methylethyl)amino)-3-(2-methylphenoxy)propan-2-ol

Constituent 2

Reference substance name:

1-((2-Amino-1-methylethyl)amino)-3-(2-methylphenoxy)propan-2-ol

Molecular formula:

C13H22N2O2

IUPAC Name:

1-((2-Amino-1-methylethyl)amino)-3-(2-methylphenoxy)propan-2-ol

Constituent 3

Reference substance name:

3,3'-(Propane-1,2-diylbis(azanediyl))bis(1-(2-methylphenoxy)propan-2-ol)

Molecular formula:

C23H34N2O4

IUPAC Name:

3,3'-(Propane-1,2-diylbis(azanediyl))bis(1-(2-methylphenoxy)propan-2-ol)

Constituent 4

Reference substance name:

1,3-Bis(2-methylphenoxy)propan-2-ol

Molecular formula:

C17H20O3

IUPAC Name:

1,3-Bis(2-methylphenoxy)propan-2-ol

Constituent 5

Reference substance name:

unknown compounds

Molecular formula:

not applicable (unknown compounds)

IUPAC Name:

unknown compounds

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

other: reconstructed human epidermis

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTMSM, EPISKIN SNC Lyon, France
Supplier: SKINETHIC Laboratories; 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 15-EKIN-050
- Expiry date: 21 December 2015
- Date of initiation of testing: 16 December 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 42 hours at 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: approximately 25 mL PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis).
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT per well
- Incubation time: 3 h at 37 °C
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: Water-killed epidermis for MTT-interaction items: The living epidermis was placed in a 12 well plate with 2 mL of distilled water (replacing the culture medium). Incubated at 37 °C, 5% CO2, for 48 hrs +/- 1 hour. At the end of the incubation, the water was discarded. Kept dead epidermis frozen (dry) in freezer at -18°C to -20°C (killed epidermis can be stored and used up to 6 months). Before use, the killed tissues were de-frozen at room temperature (app. 1 hour in 2 mL of assay medium). Further use of killed tissues was similar to living tissues.
- N. of replicates: 3
- Method of calculation used: Non specific MTT reduction calculation (NSMTT)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if the viability after 15 minutes exposure and 42 hours post incubation is less than or equal to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: Additional controls for dyes and chemicals able to color the tissue

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 µL

NEGATIVE CONTROL
- Amount applied: 10 µL
- Concentration: 1 x PBS

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5% SDS aq.

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

12.42

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

relative viability

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: During the check-method for possible direct MTT reduction, colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary. The non-specific MTT reduction (NSMTT) was determined to be 0.091%. As the NSMTT was below 30 % the true MTT metabolic conversion and the correction of viability percentages were undertaken.
- Colour interference with MTT: As the test item has an intrinsic colour (light yellow), two additional chemical-treated tissues were used for the non-specific OD evaluation. The mean OD (measured at 570 nm) of these tissues were determined as 0.031. The Non Specific Colour % (NSC %) was calculated as 3.7 %. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study, using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD value of the three negative control tissues was 0.861.
- Acceptance criteria met for positive control: Yes. The mean OD value obtained for the positive control was 0.078.
- Acceptance criteria met for variability between replicate measurements: Each calculated standard deviation value (SD) for the % viability was below 18.
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Applicant's summary and conclusion

Interpretation of results:

other: GHS Category 2 and/or Category 1

Conclusions:

According to the current OECD guideline 439, the results indicated that the the test item is irritant (Category 2) and /or corrosive (Category 1). However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion will be required to decide on its final classification.

Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. The test item acted directly on MTT (MTT-reducer), therefore additional controls (three test item treated killed tissues and three negative control killed tissues) were used to detect and correct for test substance interference with the viability measurement. As the test item has an intrinsic colour (light yellow), two additional chemical-treated tissues were used for the non-specific OD evaluation. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. The test item showed significantly reduced cell viability (corrected value) in comparison to the negative control (mean value: 12 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results indicated that the the test item is irritant (Category 2) and /or corrosive (Category 1). However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion will be required to decide on its final classification.