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Description of key information

D-C16 is non-irritant in the in vitro Episkin skin irritation test.
D-C16 did not induce ocular irritation in a BCOP test inducing an IVIS ≤ 3; therefore, no classification is required for eye irritation or serious eye damage.

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
Study period:
February - March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD method without significant deviations
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Reconstructed Human Epidermis Test Method
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: skin derived from human adults
Strain:
not specified
Details on test animals and environmental conditions:
Test system: EPISKIN Small Model (EPISKIN-SM, 0.38 cm², Batch no.: 15-EKIN-010).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results
in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Type of coverage:
other: not applicable as in vitro study
Preparation of test site:
other: not applicable as in vitro study
Controls:
other: not applicable as in vitro study
Number of animals:
not applicable as in vitro study
Details on study design:
Preparation and preincubation
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37 °C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL).
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 70 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.7 - 37.2 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Study design
A test substance may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test substance is present on the tissues when the MTT viability test is performed.
Test for colour interference by the test substance
D-C16 was checked for possible colour interference before the study was started. Some non-coloured test substances may change into coloured substances in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, approximately 10 mg of D-C16 was added to 90 µl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 µl Milli-Q water was tested concurrently. At the end of the shaking period colour check is performed. In case the solution turned blue / purple the test substance may have colour interference. A functional test with living skin tissues was performed to show that the test substance did not bind to the tissue and resulted in a false MTT reduction signal.
Test for reduction of MTT by the test substance
D-C16 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 10 mg of the test substance was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37 °C. A negative control, sterile Milli-Q water was tested concurrently.
In case the test substance reacts with the MTT medium in addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues must be used for the cytotoxicity evaluation with MTT.
Application/Treatment of the test substance
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 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 ± 0.5 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
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL). The tissues were incubated for 3 h at 37 °C. After incubation the tissues were placed on blotting paper to dry the tissues.Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
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.
Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme(s):
REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature and humidity.
Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.
Irritation / corrosion parameter:
other: other: optical density 570 nm
Value:
106
Remarks on result:
other:
Remarks:
Basis: mean of 3 experiments. Time point: 15 minutes treatment. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Negative control is set to 100% whereas mean scores for test substance were at 106%, The positive control showed 44%. (migrated information)
Irritant / corrosive response data:
D-C16 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because no colour changes were observed it was concluded that D-C16 did not interact with the MTT endpoint.

Negative control (Phosphate buffered saline (PBS)): Mean tissue viability 100%
Test substance D-C16: Mean tissue viability 106%
Positive control (5% (aq) sodium dodecyl sulphate (SDS)): Mean tissue viability 44%

The results above shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with D-C16 compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with D-C16 compared to the negative control tissues was 106%. Since the mean relative tissue viability for D-C16 was above 50% D-C16 is considered to be non-irritant. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 44%. The absolute mean OD 570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically with positive control was 27%, which was due to one outlier. However, since the other 2 viabilities were clearly positive and within the historical data range and furthermore the test substance showed a clearly negative result this did not affect study integrity. The standard deviation value of the percentage viability of three tissues treated identically with negative control or test substance was less than 8%.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
D-C16 is non-irritant in the in vitro skin irritation test.
Executive summary:

The study describes the ability of D-C16 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SM TM )). The possible skin irritation potential of D-C16 was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines.

Skin tissue was moistened with 5 µL of Milli-Q water and an excessive amount of D-C16 was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with D-C16 compared to the negative control tissues was 106%. Since the mean relative tissue viability for D-C16 was above 50% after 15 ± 0.5 minutes treatment D-C16 is considered to be non-irritant.

The positive control had a mean cell viability of 44% after 15 ± 0.5 minutes exposure. The absolute mean OD 570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically with positive control was 27%, which was due to one outlier. However, since the other 2 viabilities were clearly positive and within the historical data range and furthermore the test substance showed a clearly negative result this did not affect study integrity. The standard deviation value of the percentage viability of three tissues treated identically with negative control or test substance was less than 8%.

Finally, it is concluded that this test is valid and that D-C16 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
Study period:
February - March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD method without significant deviations
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: bovine eyes from slaughter house
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects 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-Ferrand, 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:
unchanged (no vehicle)
Controls:
other: negative controls (physiological saline) and positive controls (20% (w/v) imidazole) were used.
Amount / concentration applied:
No correction was made for the purity/composition of the test substance. Since no workable suspension of D-C16 in physiological saline could be obtained, the test substance was used as delivered by the sponsor and added pure on top of the corneas.
Duration of treatment / exposure:
not applicable, incubation of corneas was performed for 240 ±10 minutes at 32 ±1 °C.
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
3 corneas per group were used
Details on study design:
Cornea selection and Opacity reading:
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont-Ferrand, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.
Treatment of corneas and opacity measurements:
The medium from the anterior compartment was removed and 750 µl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. D-C16 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (321.7 to 358.4 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. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
Opacity measurement
The opacitometer determined the difference in the light transmission between each control or 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 change in 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.
Application of sodium fluorescein:
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ±5 minutes at 32 ±1 °C.
Permeability determinations:
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 µl 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 the dilution factor was applied to the readings.
Electronic data capture:
Observations/measurements in the study were recorded electronically using the following programme: Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.
Interpretation:
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints.
The IVIS cut-off values for identifying the test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range ≤ 3  No UN GHS Category
In vitro score range > 3; ≤ 55  No UN GHS prediction can be made
In vitro score range >55  UN GHS Category 1
Acceptability of the assay:
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean value.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
6.12. List of deviations
In the solvent control measurement, one cornea was not placed correctly in the holder and this measurement was excluded from the IVIS determination. Since the other two measurements were within the historical data, this deviation in the IVIS determination had no influence on the study results. The study integrity was not adversely affected by the deviation.
Any deviations from standard operating procedures were evaluated and filed in the study file. There were no deviations from standard operating procedures that affected the integrity of the study.
Irritation parameter:
other: in vitro irritation score (IVIS)
Basis:
mean
Score:
-0.5
Max. score:
135.4
Reversibility:
other: not applicable
Remarks on result:
other: The max. score is taken from positive control results; negative control was -0.5.
Irritant / corrosive response data:
D-C16 was tested as it is.
The table “Summary of opacity, permeability and in vitro scores “summarizes the opacity, permeability and in vitro irritancy scores of D-C16 and the controls.
The individual in vitro irritancy scores for the negative controls ranged from -1.1 to 0.1. In the solvent control measurement, one cornea was not placed correctly in the holder and was excluded from the IVIS determination. Since the other two measurements were within the historical control data base, this deviation in the IVIS score determination had no influence on the study results. The individual positive control in vitro irritancy scores ranged from 130 to 141. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with D-C16 showed opacity values ranging from -1.0 to 0.0 and permeability values ranging from 0.000 to 0.028. The corneas were clear after the 240 minutes of treatment with D-C16. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.0 to 0.4 after 240 minutes of treatment with D-C16.
Other effects:
No effect on pH was observed

Summary of opacity, permeability and in vitro scores

Treatment

Mean Opacity

Mean Permeability

Mean In vitro Irritation Score*

Negative control

-0.5

0.000

-0.5

Positive control

107.0

1.896

135.4

D-C16

-0.7

0.013

-0.5

* Calculated using the negative control mean opacity and mean permeability values. In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
D-C16 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.5 after 240 minutes of treatment.
Since D-C16 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

Evaluation of the eye hazard potential of D-C16 was performed using the Bovine Corneal Opacity and Permeability test (BCOP test, OECD 429). The report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of D-C16 was tested through topical application for approximately 240 minutes. 

Batch 14-002 of D-C16 was a white crystal with a purity of 99.3%. Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas (321.7 to 358.4 mg).

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 135 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 

D-C16 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.5 after 240 minutes of treatment. Since D-C16 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

D-C16 was tested for skin and eye irritation/damage in two in vitro tests:

In an Episkin model test no signs of irritation by the test substance were observed and thus the substance is not considered a skin irritatnt. Furthermore, in a BCOP test for eye irritation/damage, no irritation was noted and thus the substance is also not considered being an eye irritant.

Justification for classification or non-classification

As D-C16 neither induced skin nor eye irritation in two in vitro tests (Episkin and BCOP) the substance is not subject to classification and labelling for skin or eye irritation according to CLP (Regulation EC No 1272/2008).