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

Endpoint summary

Description of key information

X300 is not irritant to skin and eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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.

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

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

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

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%

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%).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 070601
- Expiration date of the lot/batch: 06/06/2019
- Purity test date: 31/07/2017
- Purity: 92,70%
- Form: solid (flakes)


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:room temperature, in a dry place

Used as supplied

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed Human epidermis

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

This test uses the reconstructed human epidermis model (Episkin RHE) which consists of normal human keratinocytes 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:RHE/S/17 de Episkin
- Tissue batch number(s): 17-RHE-094
- Production date: 12/09/2017
- Shipping date: 12/09/2017
- Delivery date: 12/09/2017
- Date of initiation of testing: 12/09/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable):37°C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 25 x 1 ml
- Observable damage in the tissue due to washing: rinsed tissues checked for any coloration and noted to be witish, comparable to negative control tissues.
- Modifications to validated SOP: none
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: ELx800 absorbance microplate reader
- Wavelength: 570 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1,2 (MTT test)
- Barrier function: ET50 = 5,1h
- Morphology: 6 cells layers, absence of significant histological abnormalities, well differentiated epidermis
- Contamination: on blood of the same donor: absence of HIV1 and 2, hepatitis C, antibodies, absence of hepatitis B antigen, on epidermal cells of the same donor: absence of mycoplasma
- Reproducibility: positive and negative controls demonstrate reproducibility in results of 2016.

NUMBER OF REPLICATE TISSUES: 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 irritant or corrosive to skin if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-corrosive to skin if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is strictly greater than 50%.

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg + 10µl (distilled water)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16µl

POSITIVE CONTROL
- Amount(s) applied (volume or weight):16 µl
- Concentration (if solution):5%

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

ca. 88.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

ca. 77.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

ca. 75.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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
- Acceptance criteria met for positive control:yes

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation EC No. 1272/2008, the test item X300 has to be considered as non-irritant to skin. It corresponds to UN GHS No Category.

Executive summary:

The aim was to evaluate the possible irritating effects of the test item X300 after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model).

The test item X330 was applied as supplied at the dose of 16 mg, during 42 minutes, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) previously moistened with 10 μL of distilled water. The application was followed by a rinse with 25 mL of DPBS and a 42 hours postincubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The experimental protocol was established in accordance with O.E.C.D. Test Guideline No.439 adopted 28 July 2015 and the Test method B.46 of Council regulation No. 761/2009 dated 23 July 2009 (EU Journal L220) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

The mean percent viability of the treated tissues was 80.5%, versus 2.4% in the positive control (5% Sodium Dodecyl Sulfate).

In accordance with the Regulation EC No. 1272/2008, the test item X300 has to be considered as non-irritant to skin. It corresponds to UN GHS No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted 26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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
- Solubility and stability of the test substance in the solvent/vehicle: stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): fresh eyes collected from the slaughterhouse on test day and transported in HBSS containing Pen/Strep on ice to the laboratories. Corneas are then incubated with RPMI (1% Fetal Bovine Serum and 2 mM L-glutamine, without phenol red)) for 1 hour at 32 +/-1 °C
- Time interval prior to initiating testing: 1 hour
- indication of any existing defects or lesions in ocular tissue samples: corneas were visually carefully examined for defects prior equilibrium storage and any defective cornea had been discarded also after a first check of opacity after equilibrium.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 20%

VEHICLE / NEGATIVE CONTROL : physiological saline NaCl 0.9%
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 0.9%
- - Lot/batch no. (if required): 1406788/B Braun

POSITIVE CONTROL
- Amount apply: 750 µl
- Name: imidazole 20% in physiological saline 0.9%
- Lot/batch no. : SLBK9670V : Sigma

Duration of treatment / exposure:

4 hours +/- 5 minutes

Duration of post- treatment incubation (in vitro):

90 min at 32 +/-1°C

Number of animals or in vitro replicates:

3 per group

Details on study design:

BCOP (= Ex Vivo pour REACH)
SELECTION AND PREPARATION OF CORNEAS
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera.
The isolated corneas were stored in a petri dish containing HBSS. After quality check step 1, the isolated corneas were mounted in corneal holders (BASF, Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32  1 °C.
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer (Quality check step 2).
QUALITY CHECK OF THE ISOLATED CORNEAS
Step 1: The cornea had been visually examined for defects and any defective cornea had been discarded.
Step 2: Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay.
NUMBER OF REPLICATES 3 for each group (test item, positive control and negative control)

NEGATIVE CONTROL USED
3 corneas treated with imidazole 20% in physiological saline 0.9% NaCl
POSITIVE CONTROL USED
3 corneas treated with imidazole 20% in physiological saline 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME
750 µl during 4 hours +/- 5 minutes at 32 +/- 1 °C
REATMENT METHOD:: closed chamber.
POST-INCUBATION PERIOD: yes/no. If YES please specify duration

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 with MEM to remove controls/test item + 1 with complete RPMI
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer (BASF-OP3.0, Duratec GmbH)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry (OD490)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS UN GHS
< or = 3 No category
> 3; < or = 55 No prediction can be made
> 55 Category 1

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. The IVIS cut-off values are identical to the ones in TG 437.

Irritation parameter:

in vitro irritation score

Value:

0.98

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

According to the evaluation criteria X300 is classified into UN GHS No Category.

Executive summary:

The eye irritancy potential of the test substance was investigated in the bovine corneal opacity and permeability assay according to the OECD guideline number 437. The test item was suspended with physiological saline 0.9% NaCl to gain a 20% concentration. The following mean in vitro irritation score was calculated: 0.98. Therefore the test item was classified into UN GHS No Category. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The corrosion assay shows that the test item is not corrosive.

The irritation test on the same test item shows that there are no irritating effects.

The test item is therefore not consider as an irritant skin substance.

The test item is not irritant for the eyes as well.