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Description of key information

In vitro skin corrosion

The test material was not corrosive in the in vitro skin corrosion test.

In vitro eye irritation

Under the conditions of the study no prediction of eye irritation can be made.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 February 2018 to 23 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model kit (EPI-200)
- Tissue lot number(s): 27958

TEST FOR MTT REDUCTION
A test material 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 material is present on the tissues when the MTT viability test is performed.
At least 25 mg of the test material or 50 µL Milli-Q water as a negative control were added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C. At the end of the exposure time it was checked if a blue / purple colour change or a blue / purple precipitate was observed.

ASSESSMENT OF COLOUR INTERFERENCE WITH MTT
The test material was checked for possible colour interference before the study was started. Approximately 25 mg of the test material or 50 µL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.

MAIN TEST

PRE-INCUBATION
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 1 hour at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM just before the test material was applied.

APPLICATION OF TEST MATERIAL AND CONTROLS
The test was performed on a total of 4 tissues per test material together with a negative control and positive control. Two tissues were used for a 3-minute exposure to test material and two for a 1-hour exposure. The skin was moistened with 25 µL Milli-Q water to ensure close contact of the test material to the tissue and 30.3 to 37.7 mg of the solid test material was added into the 6-well plates on top of the skin tissues.
For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point.

REMOVAL OF TEST MATERIAL AND CONTROLS
After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test material. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.

CELL VIABILITY MEASUREMENT
The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37 °C in air containing 5 % CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol overnight at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be < 15 %.
c) In the range 20 - 100 % viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30 %.

INTERPRETATION
A test material is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
b) In addition, a test material considered non-corrosive (viability ≥ 50 %) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15 %.
A test material is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.



Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 30.3 - 37.7 mg (with 25 µL Milli-Q water to increase tissue surface contact)

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8.0 N
Duration of treatment / exposure:
3 minutes or 60 minutes
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
100
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute expoure
Value:
70
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
DIRECT MTT REDUCTION AND ASSESSMENT OF COLOUR INTERFERENCE
The test material was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test material to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test material did not interfere with the MTT endpoint.

TEST MATERIAL AND CONTROLS
Mean OD570 values and viabilities for the negative control, positive control and test material are given in Table 1.
The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with test material compared to the negative control tissues was 100 and 70 % respectively. Because the mean relative tissue viability for the test material was not below 50 % after 3 minutes treatment and not below 15 % after 1 hour treatment the test material is considered to be not corrosive.

QUALITY CRITERIA
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.9 %. In the range of 20 - 100 % viability the Coefficient of Variation between tissue replicates was ≤ 10 %, indicating that the test system functioned properly.

Table 1: Mean OD570 Values and Viabilities for the Negative Control, Positive Control and Test Material

Tissue

Exposure period

Mean OD570 of individual tissues

Mean OD570 of duplicate tissues

Standard deviation

Coefficient of Variation (%)

Relative Mean Viability (%)

Negative control

3 minutes

1.579

1.643

0.090

7.4

100

1.706

60 minutes

1.749

1.792

0.060

4.7

1.834

Positive control

3 minutes

0.153

0.149

0.005

5.0

9.1

0.146

60 minutes

0.135

0.142

0.009

8.3

7.9

0.148

Test material

3 minutes

1.707

1.643

0.090

7.5

100

1.579

60 minutes

1.178

1.246

0.096

10

70

1.314

OD = optical density

 

Interpretation of results:
other: Not corrosive in accordance with EU criteria
Conclusions:
Under the conditions of the study, the test material was not corrosive in the in vitro skin corrosion test.
Executive summary:

The potential of the test material to cause corrosion to the skin was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.bis under GLP conditions using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

During the study, duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period.

Under the conditions of the study the positive control had a mean relative tissue viability of 7.9 % after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range.  In the range of 20 - 100 % viability the Coefficient of Variation between tissue replicates was ≤10%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material.  The relative mean tissue viability obtained after 3-minute and 1-hour treatments with test material compared to the negative control tissues was 100 and 70 %, respectively. Because the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment the test material is considered to be not corrosive.

In conclusion, the test material was not corrosive in the in vitro skin corrosion test.

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:
05 March 2018 to 27 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
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:
2017
GLP compliance:
yes
Test material information:
Composition 1
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- 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.
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 424.8 - 462.7 mg
Duration of treatment / exposure:
240 ± 10 minutes
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes with sodium fluorecein
Number of animals or in vitro replicates:
3 replicates for each condition (test material, negative control and positive control)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularisation 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 (Earle’s Minimum Essential Medium containing 1 % (v/v) L-glutamine (Life Technologies) and 1 % (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
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. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
: physiological saline

POSITIVE CONTROL USED
: 20 % (w/v) Imidazole solution prepared in physiological saline

TREATMENT METHOD
Initially, this experiment was rejected since some of the acceptability criteria were not met. A repeat experiment was performed.
The medium from the anterior compartment was removed and 750 µL of the negative control and positive control were introduced onto the epithelium of the cornea. The test material was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (424.8 to 462.7 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.

REMOVAL OF TEST SUBSTANCE
After the incubation the solutions and the test material were removed and the epithelium was washed at least three times with MEM with phenol red. Possible pH effects of the test material 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 opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity = [(I0/I) - 0.9894] / 0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
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 treated cornea with the test material or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test material or positive control 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 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. 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 has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.

DATA INTERPRETATION
The test material was classified according to the following:
- IVIS ≤ 3 = Not classified for irritation
- IVIS > 3; ≤ 55 = No prediction can be made
- IVIS > 55 = Category 1, H318: Causes serious eye damage

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.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Irritation parameter:
in vitro irritation score
Run / experiment:
Experiment 1
Value:
22
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
in vitro irritation score
Run / experiment:
Experiment 2
Value:
1.2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
TEST MATERIAL
The corneas treated with the test material showed opacity values ranging from -1.5 to 1.1 and permeability values ranging from 1.060 to 2.146. The corneas were slightly translucent after the 240 minutes of treatment with the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 17 to 32 after 240 minutes of treatment with the test material.
In the second experiment, the corneas treated with the test material showed opacity values ranging from -2.5 to -1.9 and permeability values ranging from 0.065 to 0.541. The corneas were clear after the 240 minutes of treatment with the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.0 to 5.6 after 240 minutes of treatment with the test material.

POSITIVE AND NEGATIVE CONTROLS
In the first test, the individual in vitro irritancy scores for the negative controls ranged from 1.8 to 5.3. One of the negative control eyes was excluded from the analysis since the opacity was above the historical data base which resulted in an IVIS outside the historical data base. Since the mean of the two remaining eyes completely met the criteria and the test material results were not influenced by this result, this was not considered to affect the study outcome. The individual positive control in vitro irritancy scores ranged from 162 to 166. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
Since one of the negative controls was excluded an additional test was performed.
In the second experiment, the individual in vitro irritancy scores for the negative controls ranged from 3.2 to 5.9. The individual positive control in vitro irritancy scores ranged from 111 to 146. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

Table 1: Summary of opacity, permeability and in vitro irritancy scores - Experiment 1

Treatment

Mean Opacity

Mean Permeability

Mean In Vitro Irritancy Score

Negative control

2.7

0.055

3.6

Positive control

131

2.161

164

Test material

-0.3

1.488

22

Table 2: Summary of opacity, permeability and in vitro irritancy scores - Experiment 2

Treatment

Mean Opacity

Mean Permeability

Mean In Vitro Irritancy Score

Negative control

4.6

0.021

4.9

Positive control

96

2.515

133

Test material

-2.2

0.226

1.2

Interpretation of results:
other: According to EU criteria, no prediction on eye irritation can be made
Conclusions:
Under the conditions of the study no prediction of eye irritation can be made.
Executive summary:

The potential of the test material to cause eye irritation was investigated in accordance with the standardised guideline OECD 437, under GLP conditions.

During the study eyes from slaughtered cattle were treated with 424.8 to 462.7 mg test material. Positive and negative controls were tested concurrently.

The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

In the first test, 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 (after exclusion of one outlioer).  The mean in vitro irritancy score of the positive control (20 % (w/v) imidazole) was 164 and within two standard deviations of the current historical positive control mean.  It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  

The test material induced ocular irritation through one endpoint, resulting in a mean in vitro irritancy score of 22 after 4 hours of treatment.

In conclusion, since the test material induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.  

Since one of the negative controls was excluded an additional test was performed.

In the second experiment, the mean 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 133 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The mean in vitro irritancy score was 1.2 after 240 minutes of treatment with the test material. Since the results for the test material were spread over 2 categories (-0.9, 5.6 and -1.0, respectively), no prediction about the category could be made.  

In conclusion, since the test material induced irritancy through the endpoint permeability, no prediction on the classification can be made.

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro skin corrosion

The potential of the test material to cause corrosion to the skin was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.bis under GLP conditions using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period.

Under the conditions of the study the positive control had a mean relative tissue viability of 7.9% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range.  In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤10%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material.  The relative mean tissue viability obtained after 3-minute and 1-hour treatments with test material compared to the negative control tissues was 100% and 70%, respectively. Because the mean relative tissue viability for the test material was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test material is considered to be not corrosive.

In conclusion, the test material was not corrosive in the in vitro skin corrosion test.

In vitro eye irritiation

The potential of the test material to cause eye irritation was investigated in accordance with the standardised guideline OECD 437, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study eyes from slaughtered cattle were treated with 424.8 to 462.7 mg test material. Positive and negative controls were tested concurrently.

The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

In the first test, 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 (after exclusion of one outlioer).  The mean in vitro irritancy score of the positive control (20 % (w/v) imidazole) was 164 and within two standard deviations of the current historical positive control mean.  It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  

The test material induced ocular irritation through one endpoint, resulting in a mean in vitro irritancy score of 22 after 4 hours of treatment.

In conclusion, since the test material induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.  

Since one of the negative controls was excluded an additional test was performed.

In the second experiment, the mean 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 133 and within two standard deviations of the current historical positive control mean.  It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The mean in vitro irritancy score was 1.2 after 240 minutes of treatment with the test material. Since the results for the test material were spread over 2 categories (-0.9, 5.6 and -1.0, respectively), no prediction about the category could be made.  

In conclusion, since the test material induced irritancy through the endpoint permeability, no prediction on the classification can be made.  

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin and eye corrosion or irritation.