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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 15, 2016 - October 6, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiSkin™ SOP, Version 1.8
Version / remarks:
February 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
TOXI-COOP ZRT.8230 Balatonfüred, Arácsi út 97. (1103 Budapest, Cserkesz u. 90) Hungary

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate
EC Number:
947-903-4
Cas Number:
1917323-93-3
Molecular formula:
C28H42Ca3N4O16 and C10H16CaN2O5
IUPAC Name:
Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate
Test material form:
other: highly viscous, semi-solid mass

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult human donors
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. 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: EpiSkin(TM)SM, EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model
- Tissue batch number(s): 16-EKIN-034
- Expiry date: August, 29, 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: approximately 25 mL PBS 1 x solution; rest of the PBS was removed from 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: 3 mg/mL stock solution; 2 mL of 0.3 mg/mL per well
- Incubation time: 3 hours
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates : 2

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant 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.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is greater than 50% of the negaive control.
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: The test item is a highly viscous material therefore exact weighting of treatment volume could not be performed. The test item was applied with spatula, in such a way that the entire surface of a plastic film was covered with test substance.

VEHICLE
- Amount(s) applied: 10 µL
- Lot/batch no.:SLBH9016

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration: 5% aq. solution
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1. Experiment
Value:
103
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
15%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be excluded.
- Colour interference with MTT: As the test item has an intrinsic colour, two additional test item-treated tissues were used for the non-specific OD evaluation. The mean OD of these tissues was determined as 0.075. The Non Specific Colour % (NSC %) was calculated as 12.2%. Therefore additional data calculation was necessary

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) 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 0.618.
- Acceptance criteria met for positive control: yes
The mean OD value obtained for the positive control was 0.094 and this result corresponds to 15 % viability when compared to the results obtained from the negative controls.
- Acceptance criteria met for variability between replicate measurements: yes
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:
GHS criteria not met
Conclusions:
The test item is considered as non-irritant in the EPISKIN in vitro skin irritation test.
Executive summary:

The skin irritation potential of the test item on reconstructed human epidermis in the EPISKIN model was determined according OECD TG 439 and EU method A.46.

Reconstructed human epidermis was 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 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.

As the test item has an intrinsic colour (brown), two additional test item 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 irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid