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Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

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Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of experiment: November 29th, 2016 - Completion of experiment: December 14th, 2016
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Principles of method if other than guideline:
The irritation potential of the test item was predicted by measurement of its cytotoxic effect, as reflected in the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,Thiazolyl blue tetrazolium bromide; CAS number 298-93-1] assay, on the reconstructed human epidermis model (RhE).

For every application time (3 minutes and 1 hour) the cell viability based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt was quantitatively measured after extraction from tissues. The reduction of cell viability in treated tissues was compared to negative control and expressed as a %. The % reduction in viability is used to predict the corrosion potential.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item name: Delmopinolo HCl
Batch number: 2510468
Internal number: 2929564-001
Chemical name 2-[3-(4-propylheptyl)-4-morpholinyl) ethanol hydrochloride (1:1)
Purity: 100.3% w/w
Stability under storage conditions: protect from moisture
Storage condition: The test item will be stored at room temperature without particular precaution to avoid the light exposure following Test Substance Data Sheet (TSDS) supplied by the Sponsor.

Stability under storage conditions: protect from moisture

Storage condition: The test item will be stored at room temperature without particular precaution to avoid the light exposure following Test Substance Data Sheet (TSDS) supplied by the Sponsor.
Test system:
human skin model
Source species:
human
Cell type:
other: Normal human-derived epidermal keratinocytes
Cell source:
other: Human
Source strain:
other: Not relevant because in vitro test.
Details on animal used as source of test system:
Not relevant because in vitro test.
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM reconstituted human epidermis
- Tissue batch number(s): 23382
- Expire date: December 03rd, 2016 (4 days after receipt date, according to internal POS_M_55)
- Date of initiation of testing: November 29th, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: during the test 37.6°C

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: The MTT concentrate defrozen was diluted (1:5) with MTT diluent to a final concentration of 1 mg/ml. The obtained solution was stored for later use on the same day protected from light.
- Incubation time: Exposure of test item: 3 minutes and 1 hour.
- Spectrophotometer: Spectrophotometer Biotek PowerWave XS No. 369
- Wavelength: 570 ± 30 nm

Experimental Design
Pre-incubation: Prior to start the experiment the assay medium was left to reach room temperature before use. In the meantime, the epidermis units were placed in an empty sterile 24-well plate for visual inspection. During the pre-incubation time, were prepared two 24-well plates to be used as “holding plate”: one for the 3 minutes and the other for the 1 hour application time. Each well was filled with 0.3 ml of assay medium. In addition were prepared other two 24-well plates for the MTT assay (one for 3 minutes and
one for 1 hour application time), filled with 0.3 ml of MTT solution (1mg/ml). The plates were incubate at 37 °C with 5 % CO2 until their use.

Chemical Exposure: 1 hour application time - After 1 hour of pre-incubation, each insert was transferred in new 6-well plates pre-filled with fresh assay medium (0.9 mL for each well).
Test Item
25 mg of test item undiluted was applied on the top of epidermis. After application the plate was tapped in order to cover evenly all the tissues surface.
Between each application it was waited 60 sec. The plates with the treated epidermis units were incubated at 37°C and 5% CO2 for the remaining time to 60 minutes calculating the first tissue application as the start.

Rinsing
After the incubation time, the EpiDermTM units were removed and rinsed thoroughly with DPBS solution, filling and emptying the tissue insert 20 times to remove all residual test
substance from the epidermal surface. The excess of DPBS was removed gently shaking the inserts and drying them on blotting paper. The rinsing phase was finished in 60 sec for each insert. After each rinsing, the insert was placed into the holding plate pre-filled with 0.3 ml of assay medium until also the last tissue was washed. At these time, each insert was dried on the surface with cotton swab.

MTT Viability Test
At this time all tissues were ready to be transferred in the MTT plate pre-filled before with 0.3
ml MTT solution and then incubated for 3 hours at 37°C and 5 % CO2.

Chemical Exposure: 3 minutes application time
Test Substances Application - Due to the short application time, each couple of tissue was applied, rinsed, dried and placed in holding plate before starting with the other two tissues.

MTT Viability Test and Formazan extraction
When all the tissues were rinsed and positioned in the holding plate, then they were ready to be transferred in the MTT plate pre-filled before with 0.3 ml MTT solution and then incubated for 3 hours at 37°C and 5 % CO2. At the end of incubation with MTT, a formazan extraction was undertaken both for 1 hour and 3 minutes application time: Starting from 1 hour the MTT medium was aspirated from the wells, refilled and emptied three times with DPBS, then the tissues were transferred to new 24-well plates for 1 hour application time. Than the inserts were immersed pipetting 2 ml of extractant solution and the 24-well plate was sealed with Parafilm to inhibit extractant evaporation. Same way of operating was done for 3 minutes application time. Each 24-well plates were left at room temperature protected from light overnight. After extraction, all inserts were pierced with a pipette tip, in order to recover the extract in the wells. At this time, the inserts were discarded.

Cell viability measurements
Following the formazan extraction, the plates were shook at 120 rpm for 15 minutes until the solution was homogeneous in colour. After that, 2 x 200 μL aliquots from each well were placed into the wells of a 96-well plate and the OD (Absorbance / Optical Density) of the samples was read in a 96-well plate spectrophotometer at the wavelength of 570 ± 30 nm using isopropanol as the blank (6 × 200 μL).

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if 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%.

Number of replicate wells
In this assay 2 replicates per test item; 2 replicates negative controls, 2 replicates positive controls were used for each application time.


Control samples:
other: yes, concurrent MTT - positive control (KOH 8 N) and negative control (Sterile demineralised water (filtered on 0.22 μm))
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of the test item was added to 0.3 ml of deionized water

NEGATIVE CONTROL/ POSITIVE CONTROL
- Amount(s) applied (volume or weight): A volume of 50 μl positive control (KOH 8N), and 50 μl negative control (sterile demineralized
water)
Duration of treatment / exposure:
Exposure of test item: 3 minutes and 1 hour
Duration of post-treatment incubation (if applicable):
3 hours at 37°C
Number of replicates:
2 replicates per test item; 2 replicates negative controls, 2 replicates positive controls were used for each application time.
Species:
other: In vitro test
Strain:
not specified
Remarks:
In vitro test
Details on test animals and environmental conditions:
Not relevant because an in vitro test is performed.
Type of coverage:
other: Not relevant
Preparation of test site:
other: Not relevant
Remarks:
Not relevant
Vehicle:
other: Not relevant
Remarks:
Not relevant
Controls:
other: Not relevant
Amount / concentration applied:
Not relevant because an in vitro test is performed.
Duration of treatment / exposure:
Not relevant because an in vitro test is performed.
Observation period:
Not relevant because an in vitro test is performed.
Number of animals:
Not relevant because an in vitro test is performed.
Details on study design:
Not relevant because an in vitro test is performed.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes
Value:
ca. 68.6
Vehicle controls validity:
not examined
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
The mean viability of tissues exposed to the test item was 68.6 % after 3 minutes and 10.5 % after 1 hour of the mean negative control value
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hours
Value:
ca. 10.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
The mean viability of tissues exposed to the test item was 68.6 % after 3 minutes and 10.5 % after 1 hour of the mean negative control value
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Tissue viability met the acceptance criterion because the mean OD of the Negative Controls was ≥ 0.8 and ≤ 2.8 (1.608 at 3 minutes application time and 1.565 at 1 hour application time).
- Acceptance criteria met for positive control: One hour exposure of the Positive Control showed a mean tissue viability less than 15 %.(5.0 % at 1 hour application time).
- Acceptance criteria met for variability between replicate measurements: In the range between 20 % and 100% viability, the coefficient of variation (CV) should not exceed 0.3. In this case, the maximum CV was obtained at 3 minutes application time for the test item (0.07), so the acceptance criterion was satisfied.

Interpretation of test results
Mean tissue viability (expressed as % of negative control)
STEP 1
3 min < 50 % - Corrosive
3 min ≥ 50 % AND 1 hour < 15 % - Corrosive
3 min ≥ 50 % AND 1 hour ≤ 15 % - Non-corrosive

STEP 2 - for substances identified as Corrosive in step 1
3 min. < 25% - Optional Sub-category 1A
3 min. ≥ 25 % - A combination of optional Sub-categories 1B and 1C
Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
Test item showed a strong reduced potential of cell viability in comparison to the negative control only after 1 hour application time. The mean viability of test item’s two replicates
results was 68.6% of the mean negative control value at 3 minutes application time and 10.5% at 1 hour application time. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid and qualified. In this in vitro skin corrosion test with the EpiDermTM model on test item
“DELMOPINOLO HCl” results indicated that the test item is Corrosive.
Executive summary:

In this in vitro skin corrosion test with the EpiDermTM model on test item “DELMOPINOLO HCl” results indicated that the test item is Corrosive [Requiring classification, Category 1B/1C].

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of experiment: October 03, 2016 - Completion of experimental phase: October 21, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Principles of method if other than guideline:
The irritation potential of the test item was predicted by measurement of its cytotoxic effect, as reflected in the MTT [3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS number 298-93-1] assay, on the reconstructed human epidermis model (RhE).

The cell viability based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of cell viability in treated tissues was compared to negative control and expressed as a %. The % reduction in viability is used to predict the irritation potential.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item name: Delmopinolo HCl
Batch number: 2510468
Internal number: 2929564-001
Chemical name 2-[3-(4-propylheptyl)-4-morpholinyl) ethanol hydrochloride (1:1)
Purity: 100.3% w/w
Stability under storage conditions: protect from moisture
Storage condition: The test item will be stored at room temperature without particular precaution to avoid the light exposure following Test Substance Data Sheet (TSDS) supplied by the Sponsor.

Stability under storage conditions: protect from moisture

Storage condition: The test item will be stored at room temperature without particular precaution to avoid the light exposure following Test Substance Data Sheet (TSDS) supplied by the Sponsor.
Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: Normal human-derived epidermal keratinocytes
Source strain:
other: Human
Details on animal used as source of test system:
Not relevant because in vitro test.
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM reconstituted human epidermis.
- Tissue batch number(s): 23361
- Expiry date: October 08, 2016 (4 days after receipt date, according to internal POS_M_54)
- Date of initiation of testing: October 03, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: the assay medium supplied with the kit was left at room temperature (20-25°C).
- Temperature of post-treatment incubation (if applicable): 37 °C, 5 % CO2, 95 % RH

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: The MTT concentrate defrozen was diluted (1:5) with MTT diluent to a final concentration of 1 mg/mL. The obtained solution was stored and used on the same day, at 2°C-8°C and protected from light.
- Incubation time: incubated for 1 hour at 37 °C, 5 % CO2, 95 % RH.
- Spectrophotometer: yes
- Wavelength: 570 ± 30 nm

EXPERIMENTAL DESIGN

Day 0 - Pre-incubation: The epidermis units were placed in an empty sterile 24-well plate for visual inspection.

Day 1- Chemical Exposure:
Test Item
25 mg of test item was applied on the skin surface. After application the plate was tapped in order to cover evenly all the tissues surface. Between each application it was waited 60 sec.

Post-incubation: After rinsing the units were placed into the plate wells with fresh assay medium (0.9 ml/well) and dried with the sterile cotton swab, then the plates were incubated for 24 hours (± 2) at 37 °C in an incubator with 5 % CO2, 95 % RH.

Day 2 - Change medium: After incubation period, the inserts were transferred from upper row into lower row of the 6-well plates, pre-filled with 0.9 ml of fresh assay medium and incubated at 37 °C in an incubator with 5 % CO2, 95 % RH for an additional 18 ± 2 hours.

Day 3 - MTT test after 42 hours incubation: After the 42 hours incubation the EpiDermTM units were transferred into the 24-wells plate pre filled with MTT solution (0.3 ml of 1 mg/ml MTT per well) and then incubated for 3 hours (± 5 min) at 37 °C in an incubator with 5 % CO2, 95 % RH

Formazan extraction and cell viability measurements: At the end of incubation with MTT a formazan extraction was undertaken.Following the formazan extraction, 2 x 200 μl aliquots from each well were placed into the wells of a 96-well plate and the OD (Absorbance / Optical Density) of the samples were read in a 96-well plate spectrophotometer at the wavelength of 570 ± 30 nm using isopropanol as the blank (6 × 200 μl).

Number of replicate wells:
In this assay 3 replicates per test item; 3 replicates negative control and 3 replicates positive controls were used.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
Validity of the controls: Positive control (Sodium dodecyl sulphate 5 % aq.) mean viability should be ≤ 20 % of the negative control tissues.
The mean OD value of the three negative control tissues (DPBS) should be ≥ 1.0 e ≤ 2.5.
For all treated group the standard deviation value (SD) of the % viability should be < 18 %.

Results expression: The test item is considered at least irritant to skin, if the mean relative viability is ≤ 50 % of the negative control.
Control samples:
other: yes, concurrent MTT - positive control (Sodium Dodecyl Sulphate 5 % aq.) and negative control (a phosphate buffered saline, DPBS)
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of test item was applied on the skin surface


NEGATIVE/POSITIVE CONTROL
- Concentration (if solution): A volume of 30 μl positive control (Sodium Dodecyl Sulphate SDS 5 % aq.) and negative control (a phosphate buffered
saline, DPBS) were applied on the skin surface by using a suitable pipette.
Duration of treatment / exposure:
Exposure of test item: 60 minutes.
Duration of post-treatment incubation (if applicable):
Post incubation: 42 hours.
Number of replicates:
3 replicates per test item; 3 replicates negative control and 3 replicates positive controls
Species:
other: In vitro test
Strain:
not specified
Remarks:
In vitro test
Details on test animals and environmental conditions:
Not relevant because an in vitro test is performed.
Type of coverage:
other: Not relevant
Preparation of test site:
other: Not relevant
Remarks:
Not relevant
Vehicle:
other: Not relevant
Remarks:
Not relevant
Controls:
other: Not relevant
Amount / concentration applied:
Not relevant because an in vitro test is performed.

Duration of treatment / exposure:
Not relevant because an in vitro test is performed.
Observation period:
Not relevant because an in vitro test is performed.
Number of animals:
Not relevant because an in vitro test is performed.
Details on study design:
Not relevant because an in vitro test is performed.
Irritation / corrosion parameter:
% tissue viability
Value:
ca. 5.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The mean viability of test item’s three replicates results was 5.8 % of the mean negative control value
Remarks:
The mean viability of test item’s three replicates results was 5.8 % of the mean negative control value
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:the mean OD value of the three negative control tissues should be ≥ 1.0 e ≤ 2.5
- Acceptance criteria met for positive control: the acceptable mean percentage viability range for positive controls is ≤ 20 % of the negative control tissues
- Acceptance criteria met for variability between replicate measurements:ror test item and controls, the standard deviation value (SD) of the % viability should
be < 18 %.

Criteria for In Vitro interpretation:
- Mean tissue viability % is ≤ 50 % - Requiring classification, Category 1/2 (CLP and UN GHS)
- Mean tissue viability % is > 50 % - Non Irritant, No Category (CLP and UN GHS)
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Test item showed a strong reduced cell viability in comparison to the negative control. The mean viability of test item’s three replicates results was 5.8 % of the mean negative control
value. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.
In this in vitro skin Irritation test with the EpiDermTM model on the test item “DELMOPINOLO HCl”, results indicated that the test item is at least Irritant [Category 2].
Since the Skin irritation RhE test methods cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion will be recommended to decide on test item final
classification.
Executive summary:

In this in vitro skin irritation test with the EpiDermTM model on test item “DELMOPINOLO HCl” results indicated that the test item is at least Irritant [Requiring classification, Category 2].

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study need not be conducted because the available information indicates that the criteria are met for classification as corrosive to the skin or irritating to eyes
Endpoint conclusion
Endpoint conclusion:
no study available

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Delmopinolo HCl is classified as Skin Corr. 1; H314 according to in vitro skin corrosion test with the EpiDermTM model.