Aluminate(2),[[N,N'-1,2-ethanediylbis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON]hydroxy-,disodium

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Method B.40 bis In vitro skin corrosion: human skin model test (Annex to Regulation (EC) No 440/2008)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: flakes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:040801#
- Expiration date of the lot/batch: 05.08.2016
- Purity test date:07/10/2015
- purity: 91.61%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable in water, instable after repeated contact to air


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: moistened with 100 +/- 5 µl of 0.9% NaCl solution, to ensure good contact surface.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Episkin Small Model

Source strain:

not specified

Details on animal used as source of test system:

Not applicable

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM (SkinEthic)
- Tissue batch number(s): 16-EKIN-002
- Production date: 12/01/2016
- Shipping date: 12/01/2016
- Delivery date: 12/01/2016
- Experimental starting date: 13/01/2016
- Expiration date: 18/01/2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 +/-1°C (3 hours MTT incubation)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 ml PBS, 15 times
- Observable damage in the tissue due to washing :
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/ml MTT in PBS (stock solution) / 0.3 mg/ml (1:9 DMEM-based medium (MTT medium)
- Incubation time: 3 hours
- Spectrophotometer: Plate spectrophotometer
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: absolute OD570 for blank was 0.042 (mean of 2 aliquots) for these experiment.
historical data absolute OD570 for blank was 0.044 (SD 0.002 / n=21) / Relative viability PC = 5.0 % (SD 1.9, n=21) and a maximal difference viability of 8% (SD = 7.4, n=197). Historical control data were generated in 2014-2015.
- Barrier function: IC50 = 2mg/ml (SDS concentration, MTT test, n=14 – specification ≥ 1.5 mg/ml)
- Morphology: histology scoring = 21.8 +/- 0.3, CV = 1.3% (HSE stained vertical paraffin sections, n=6 – specification ≥ 19.5)
- Contamination: Free of bacteria, fungus and mycoplasma

NUMBER OF REPLICATE TISSUES: each dose group in duplicate (6 tissues/test item)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No MTT direct interference notified
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 35%, or if the viability after 3 minutes exposure is greater than or equal to 35 % and the viability after 60 min exposure is less than 35% or if the viability after 60 minutes exposure is greater or equal to 35% and the viability after 4 hours exposure is less than 35%.]
- The test substance is considered to be non-corrosive to skin if the viability after 4 hours treatment is greater than or equal to 35%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):20 mg + 100 µl 0.9% NaCl solution

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):50 µl
- Concentration (if solution): 0.9%

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

Duration of treatment / exposure:

3 minutes / 60 minutes / 4 hours

Duration of post-treatment incubation (if applicable):

3 hours in MTT medium

Number of replicates:

Test item: 2 for each times of exposure

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean relative value

Run / experiment:

3 min

Value:

ca. 96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean relative value

Run / experiment:

60 min

Value:

ca. 103

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean relative value

Run / experiment:

4 hours

Value:

ca. 112

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

mean relative tissue viability of 3%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:Yes (Mean Absolute OD(570 nm), 3 min exp. = 0.885, Mean Absolute OD(570 nm), 60 min exp. = 0.865, Mean Absolute OD(570 nm), 4 hours exp. = 0.774). Mean absolute OD (570 nm) of the two negative control tissues of every treatment period is between 0.6 and 1.5
- Acceptance criteria met for positive control:Yes (4h exp. : 3%). Mean relative tissue viability of the 2 positive control tissues of the 4 hours treatment period is less or equal to 20%
- Acceptance criteria met for variability between replicate measurements:Yes (value 0.2 - 11.5%). In the range of 20-100% viability and for ODs superior to 0.3, the difference between each two replicates must be less or equal to 30%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

According to the in vitro skin corrosion test on a human skin model (EPISKIN-SM), following the EC guideline Method B.40 BIS, the test substance is classified as no-corrosive.

Executive summary:

The potential of the test item to induce skin corrosion was analysed by using the three-dimensional human skin model EPISKIN-SM™, comprising a reconstructed epidermis with a functional stratum corneum. In the present study the substance was applied topically to the EPISKIN-SM™ tissue for 3 min, 60 min and 4 h followed by immediate determination of cytotoxic effects via MTT reduction assay. Corrosivity potential of the test item was predicted from the relative mean tissue viabilities compared to the corresponding negative control tissues concurrently treated with 0.9% NaCl. The test item showed no non-specific MTT-reducing or water-colouring potential. The test item is no MTT-reducer and has no coloring potential, therefore no additional controls were necessary. The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 35% (112%) after 4 h treatment. Relative mean tissue viability was 103% after 60 min treatment and 96% after 3 min treatment. The controls confirmed the validity of the study. The mean OD (570 nm) of the two negative control tissues was between 0.6 and 1.5 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3%) after 4 h treatment. The maximum inter tissue viability difference of replicate tissues of all dose groups was ≤ 30% (0.2% - 11.5%).