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Description of key information

  • 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 X330 is classified as no-corrosive.

  • X330 was tested according to 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). Under these experimental conditions, the test item X330 is considered not irritating.
  • The eye irritancy potential of X330 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: 1.41. Therefore the test item was classified into UN GHS No Category.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
from 26 Septembre 2014
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:040802#
- Expiration date of the lot/batch: 06.08.2016
- Purity test date:08/01/2015
- Purity: 99.17%

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 SM
Source strain:
other: 09-KERA-009, 08-KERA-008, 08-KERA-001, 10-KERA-005
Details on animal used as source of test system:
2nd passage
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-004
- Production date: 26 January 2016
- Shipping date:26 January 2016
- Delivery date:26 January 2016
- Date of initiation of testing: 05/01/2016
- Experimental Starting date: 26 January 2016
- Experimental Completion date: 28 January 2016


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable):37 +/- 1°C, 5%CO2/ 95% air (incubator)

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

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/ml in phosphate buffered saline (PBS) (stock solution) : 0.3 mg/ml (1:9 DMEM-based medium (MTT medium)
- Incubation time: 3 hours +/- 15 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 Nm
- Filter:
- Filter bandwidth:
- Linear OD range of spectrophotometer:

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
- Reproducibility:

NUMBER OF REPLICATE TISSUES: duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No MTT direct interference notified during the pretest
- 2 killed tissues/exposure periods
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates : 2
- 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 [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 +/-2 mg +100 +/-5 µl NaCl 0.9%

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution):
Duration of treatment / exposure:
3 min, 60 min and 4 hours
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2 replicates for each exposure time for the test item and the negative control
Vehicle:
unchanged (no vehicle)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min Experiment
Value:
106
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min Experiment
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
4 hours experiment
Value:
98
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: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES for each experiments. 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 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: 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 colouring potential, therefore no additional controls were necessary. The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 35% (98%) after 4 h treatment. Relative mean tissue viability was 100% after 60 min treatment and 106% after 3 min treatment. The controls confirmed the validity of the study. The mean OD570 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% (4%) after 4 h treatment. The maximum inter tissue viability difference of replicate tissues of all dose groups was ≤ 30% (1.0% - 14.9%).

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 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:
from regulation n°761/2009
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:070602
- Expiration date of the lot/batch:06/06/2019
- Purity test date: 31/07/2017

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
- Treatment of test material prior to testing: applied as supplied.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Reconstructed Human Epidermis
Source strain:
other: PK2 PKP.AO-11
Details on animal used as source of test system:
not applicable
Justification for test system used:
This test uses the EPISKIN-RHE reconstructed human epidermis model (EPISKIN) 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 RHE/S/17
- Tissue batch number(s): 17-RHE-094
- Production date: 12 Sep. 2017
- Shipping date: 12 Sep. 2017
- Delivery date: 12 Sep. 2017
- Date of initiation of testing: 12 Sep. 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:
TTEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg + 10 µl distilled water

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

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:
95.9
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:
73.2
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:
72.4
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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean OD = 0.841
- Acceptance criteria met for positive control: mean OD = 0.02
Interpretation of results:
GHS criteria not met
Conclusions:
In accordance with the Regulation EC No. 1272/2008, the test item X330 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 X330 after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model).

The test item 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 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
Study period:
2015-2016
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: 040802#
- Expiration date of the lot/batch: 06.08.2016
- Purity test date: 08/01/2015
- Purity: 99.17%

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel:suspended with physiological saline 0.9% NaCl to gain a 20% concentration


FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid
Species:
cattle
Strain:
not specified
Remarks:
beef cattle
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 positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):750 µl
- Concentration (if solution): 20% in NaCl 0.9%

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:
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 per group

NEGATIVE CONTROL USED
3 corneas treated with 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
TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes/no. If YES please specify duration

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
Irritation parameter:
in vitro irritation score
Value:
ca. 1.41
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: (opacity and permeability values < to the established upper limits for background bovine corneas) YES
- Acceptance criteria met for positive control:(within the 2 stndard deviations of the current historical mean, within 97.2 and 161.84 in vitro irritation score)
- Range of historical values if different from the ones specified in the test guideline:within 97.2 and 161.84 in vitro irritation score
Interpretation of results:
GHS criteria not met
Conclusions:
The eye irritancy potential of X330 was investigated in the bovine corneal opacity and permeability assay.
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: 1.41
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.
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: 1.41. 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

Additional information

Justification for classification or non-classification

According to results of the 3 tests, the test item is not irritating to skin and eye.

In accordance with the Regulation EC No. 1272/2008, the test item X330 has to be considered as non-irritant to skin. It corresponds to UN GHS No Category.