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Administrative data

Description of key information

skin irritation: positive in vitro (Episkin), classified as skin irritant
eye damage/irritation: negative in vitro (BCOP), non corrosive to eyes

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:
March to May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Valid GLP guideline study without deviations
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
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 (incl. QA statement)
Species:
other: EPISKIN Small Model (EPISKIN-SM, 0.38 cm2, Batch no.1 13-EKIN-011)
Strain:
other: adult human-derived epidermal keratinocytes
Details on test animals or test system and environmental conditions:
EPISKIN Small Model ™ (EPISKIN-SM™, 0.38 cm2, Batch no.1 13-EKIN-011, received from SkinEthic Laboratories, Lyon, France). 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.
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 63 - 98%), containing 5.0 ±0.5% CO2 in air in the dark at 37.0 ±1.0 °C (actual range 36.7 - 37.5 °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 (with a maximum of 17%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
water
Controls:
other: negative and positive controls were applied
Amount / concentration applied:
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 (10.9 to 11.5 mg; with a small glass weight boat) 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.
Duration of treatment / exposure:
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 lnfinite® M200 Pro Plate Reader.
Observation period:
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.
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.
Number of animals:
All measurements (negative control, positive control and test substance) were performed in triplicates and results reported as mean
Details on study design:
Negative control: sterile Milli-Q water
Positive control: 5% aqueous sodium dodecyl sulphate solution (Sigma Aldrich) in phosphate buffered saline (PBS, Invitrogen Corp.)
Irritation / corrosion parameter:
other: other: viability
Value:
40
Remarks on result:
other:
Remarks:
Basis: mean of triplicates. Time point: following 15 minutes exposure. Max. score: 16.0. Reversibility: other: not applicable. Remarks: The max-score 16% is the viability observed with positive control. (migrated information)
Irritant / corrosive response data:
Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with BisP-IOTD compared to the negative control tissues was 40%. Since the mean relative tissue viability for BisP-IOTD was below 50% after 15 minutes treatment it is considered to be irritant. The positive control scored 16% in this test.
Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this Reconstructed Human Epidermis Test Method assay the test substance BisP-IOTD was found being irritant to skin.
Executive summary:

BisP-IOTD was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that BisP-IOTD did not interact with MTT.

The mean absorption at 570 nm measured after treatment with BisP-IOTD and controls are presented as follows (mean value of double measurements of triplicate test sample):

Negative control: OD570 = 1.074 ±0.064 (set to 100% viability)

BisP-IOTD: OD570 = 0.432 ±0.087 (i.e. 40% of negative control)

Positive control: OD570 = 0.177 ±0.069 (16% of negative control)

Mean tissue viability obtained after 15 minutes treatment with BisP-IOTD compared to the negative control tissues is provided above. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with BisP-IOTD compared to the negative control tissues was 40%. Since the mean relative tissue viability for BisP-IOTD was below 50% it is considered to be irritant.

The positive control had a mean cell viability after 15 minutes exposure of 16%. The absolute mean OD570 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 was less than 9%, indicating that the test system functioned properly.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
March to May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: fully compliant GLP guideline study without deviations
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 (incl. QA statement)
Species:
other: bovine eyes
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System: Bovine eyes were used as soon as possible after slaughter on the same day.
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, ‘s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded. The isolated corneas were stored at 32 ±1 °C in a petri dish with cMEM (Eagle's Minimum Essential Medium (lnvitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (lnvitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (lnvitrogen 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:
unchanged (no vehicle)
Remarks:
Since no workable suspension of BisP-IOTD in physiological saline could be obtained, the test substance was used as delivered by the sponsor and added pure on top of the corneas.
Controls:
other: positive and negative controls applied
Amount / concentration applied:
The medium from the anterior compartment was removed and 750 µ l of the negative control and positive control were introduced onto the epithelium of the cornea. BisP-IOTD was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (302 to 312 mg, first experiment and 345 to 355 mg, second experiment).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, lnvitrogen 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.
Duration of treatment / exposure:
Corneas were incubated in a horizontal position for 240 ±10 minutes at 32 ±1 °C.
Observation period (in vivo):
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, lnvitrogen 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.
Number of animals or in vitro replicates:
In total 18 bovine eyes were used (2 experiments with each 3 for negative control, 3 for positive control and 3 for test substance measurement)
Details on study design:
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 lnfinite® 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.
Irritation parameter:
other: opacity
Basis:
mean
Score:
4
Max. score:
135
Reversibility:
other: not applicable
Remarks on result:
other: Experiment 1 (max score is value for positive control)
Irritation parameter:
other: opacity
Basis:
mean
Score:
3
Max. score:
87
Reversibility:
other: not applicable
Remarks on result:
other: Experiment 2 (max score is value for positive control)
Irritation parameter:
other: permeability
Basis:
mean
Score:
3 790
Max. score:
2 544
Reversibility:
other: not applicable
Remarks on result:
other: Experiment 1 (max score is value for positive control)
Irritation parameter:
other: permeability
Basis:
mean
Score:
2 338
Max. score:
3 300
Reversibility:
other: not applicable
Remarks on result:
other: Experiment 2 (max score is value for positive control)
Irritation parameter:
other: in vitro irritation score (IVIS)
Basis:
mean
Score:
61
Max. score:
173
Reversibility:
other: not applicabe
Remarks on result:
other: Experiment 1 (max score is value for positive control)
Irritation parameter:
other: in vitro irritation score (IVIS)
Basis:
mean
Score:
38
Max. score:
137
Reversibility:
other: not applicable
Remarks on result:
other: Experiment 2 (max score is value for positive control)

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 (lmidazole) were 173 and 137 in the first and second experiment respectively and were within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

BisP-IOTD induced severe ocular irritation through one endpoint (permeability) only, resulting in a mean in vitro irritancy score (IVIS) of 61 after 240 minutes of treatment. In the second experiment, BisP-IOTD induced again ocular irritation through one endpoint (permeability), however the mean in vitro irritancy score was 38 after 240 minutes of treatment with BisP-IOTD. Since in vitro irritancy score for 5 out of 6 corneas treated with BisP-IOTD was below 55.1 after 240 minutes treatment, the IVIS of 84 observed in one cornea is judged as an outlaying value and therefore this increase is considered to be not relevant. Moreover the OD.,9° before the dilution step of this outlaying cornea was comparable with the other two corneas (results not reported), but after the dilution step to bring the OD into the acceptable range for measurement an increase in the OD490 was observed in this cornea.

Since BisP-IOTD induced an IVIS below 55.1 in 5 out of 6 corneas, it is concluded that BisP-IOTD is not corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Interpretation of results:
other: not corrosive or severe irritant
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this test, BisP-IOTD was found not being corrosive and thus is not classified for eye damage. However, a final decision upon eye irriation is not possible based on the BCOP test model and thus this will have to be assessed in vivo, once the next tonnage level is reached.
Executive summary:

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 (lmidazole) were 173 and 137 in the first and second experiment respectively and were within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

BisP-IOTD induced severe ocular irritation through one endpoint (permeability) only, resulting in a mean in vitro irritancy score (IVIS) of 61 after 240 minutes of treatment. In the second experiment, BisP-IOTD induced again ocular irritation through one endpoint (permeability), however the mean in vitro irritancy score was 38 after 240 minutes of treatment with BisP-IOTD. Since in vitro irritancy score for 5 out of 6 corneas treated with BisP-IOTD was below 55.1 after 240 minutes treatment, the IVIS of 84 observed in one cornea is judged as an outlaying value and therefore this increase is considered to be not relevant. Moreover the OD.,9° before the dilution step of this outlaying cornea was comparable with the other two corneas (results not reported), but after the dilution step to bring the OD into the acceptable range for measurement an increase in the OD490 was observed in this cornea.

Since BisP-IOTD induced an IVIS below 55.1 in 5 out of 6 corneas, it is concluded that BisP-IOTD is not corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The substance was investigated for skin and eye irritation/corrosion in vitro. Whereas the in vitro skin corrosion/irritation study according to the EPIskin model resulted in a classification as a skin irritant, the in vitro eye corrosion/irritation model investigatin bovine corneal opacity and permeability (BCOP) was found negative. However, it has to be considered that the BCOP model does not allow identification of eye irritating substances but only of substance causig irreversible famage to eyes (corrosive effects according to DSD). Thus, the result of the BCOP test showed that the substance is not corrosive to eyes but cannot be used to decide whether the substanc e is an eye irritant.


Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

Based on the outcome of an in vitro test for skin corrosion/irritation the substance is considered being a skin irritant category 2 according to CLP (Regulation EC No 1272/2008) respectively is classified as Xi, R36 according to DSD (Directive 67/548/EEC).

Based on the eye corrosion in vitro test using the BCOP model, the substance is not classified for irreversible effect / eye corrosion according to CLP or DSD respectively. However, as this in vitro model is not capable for identifying eye irritants, for the entire endpoint "data lacking" is appropriate.