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Administrative data

Description of key information

In vitro skin irritation:

The results obtained from an in vitro skin irritation test (EPISKIN test) indicated that the test item revealed no skin irritation potential under the utilised testing conditions.

In vitro eye corrosion:

The results obtained from an in vitro eye corrosion test indicated that the test item revealed no serious eye damaging potential under the utilised testing conditions.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin 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:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Batch: CDC-003
Species:
other: EPISKIN Small model (Human)
Details on test animals or test system and environmental conditions:
ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment.
-Humid atmosphere of 80 - 100% (actual range 69 - 87%), containing 5.0 ± 0.5% CO2 in air in the dark.
-Temperature: 37.0 ± 1.0°C (actual range 36.0 – 36.9°C).
Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
Temporary deviations from the humidity were caused by opening of the incubator door. Based on HCD, these deviations were not considered to have affected the integrity of the study.
Vehicle:
physiological saline
Amount / concentration applied:
11.5 to 17.5 mg
Duration of treatment / exposure:
15 minutes
Details on study design:
TEST SYSTEM
Name: EPISKIN Small ModelTM (EPISKIN-SM(TM), 0.38 cm2, Batch no.: 14-EKIN-024)
Source: SkinEthic Laboratories, Lyon, France.
Rationale: One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD, EC and
validated by ECVAM).

TREATMENT
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 μl
Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance (11.5 to
17.5 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at
room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts
were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

CELL VIABILITY MEASUREMENT
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 times
Value:
98
Vehicle controls validity:
valid
Remarks:
100
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
11
Remarks on result:
no indication of irritation

Mean Tissue Viability in the in vitro skin irritation test

  Mean tissue viability (percentage of control)            
Negative control 100
Test substance 98
Positive control 11
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
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 Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Batch: CDC-003
Species:
other: Bovine Eyes
Details on test animals or tissues and environmental conditions:
Preparation of corneas:
All eyes were carefully examined for defects by holding the eyes to the light after submerging in physiological saline. Those exhibiting unacceptable
defects, such as opacity, scratches, pigmentation and neovascularization were discarded.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands)
containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)).
The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the
O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws.
The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
Amount / concentration applied:
324.2 to 333.9 mg (applied directly onto corneas in such a way that the corneas were completely covered).
Duration of treatment / exposure:
240 ± 10 minutes at 32 ± 1°C
Number of animals or in vitro replicates:
Three eyes for the test substance, negative control and positive control substance.
Details on study design:
Details on treatment of corneas and opacity measurements:
The medium from the anterior compartment was removed and 750 ul of the negative control and 20% (w/v) Imidazole solution (positive control) were
introduced onto the epithelium of the cornea. The test substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (324.2 to 333.9 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the
incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and
the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the
completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

Opacity measurements:
The opacitometer determined the difference in the light transmission between each control or test substance treated cornea and an air filled chamber.The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative
control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive
control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the changein opacity of each positive control or test substance treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Permeability determination:
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled
according to holder number. 360 ul of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm
(OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was
1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the
experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using
cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before thedilution factor was applied to the readings.



Irritation parameter:
in vitro irritation score
Run / experiment:
3 times
Value:
-0.7
Vehicle controls validity:
valid
Remarks:
0.0
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
137
Irritant / corrosive response data:
The individual in vitro irritancy scores for the negative controls were -1 to 0.0. The individual positive control in vitro irritancy scores ranged from
107 to 161. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with the test substance showed opacity values of -1 and permeability values ranging from 0.002 to 0.047. The corneas were clear after the 240 minutes of treatment. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy
scores ranged from -1.0 to -0.3 after 240 minutes of treatment.

Treatment Mean Opacity Mean Permeability Mean In Vitro Irritation Score
Negative Control 0 0.000 0.0
Positive Control 92 2.986 137
Test substance -1 0.018 -0.7
Interpretation of results:
GHS criteria not met
Conclusions:
Since the test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro skin irritation: The results obtained from an in vitro skin irritation test indicated that the test item revealed no skin irritation potential under the utilised testing conditions.

In vitro eye irritation/corrosion: The results obtained from an in vitro eye irritation/corrosion test (BCOP) indicated that the test item revealed no serious eye damaging potential under the utilised testing conditions.

Justification for selection of skin irritation / corrosion endpoint: 

No indication of irritation was observed

Justification for selection of eye irritation endpoint: 

No indication of severe eye damage was observed

Justification for classification or non-classification

Based on the available experimental data the test item is not classified with regards to skin irritation/corrosion and eye irritation according to Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EC) No 2019/521.