Registration Dossier

Administrative data

Description of key information

Skin corrosion, in vitro: non-corrosive (3 -minutes viability 112% ; 1 -hour viability 103%), EPIDERM, OECD TG 431, 2017

Skin irritation, in vitro: non-irritating (15minutes exposure/42hour incubation viability = 124%), EPISKIN, OECD TG 439, 2018

Eye irritation, in vitro: non-irritating (IVIS = 0.2), BCOP, OECD TG 437, 2017

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 59%) which does not affect the reliability of the study
Qualifier:
according to
Guideline:
other: EU Method B.40 BIS : In Vitro Skin Corrosion: Human Skin Model Test
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 59%) which does not affect the reliability of the study
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm Skin Model (EPI-200, Lot no.: 22226 kit N). The test consists of topical application of the test item on the skin tissue for 3-minute and 1-hour. After exposure the skin tissue is thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The EpiDerm Skin Model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Preincubation:
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM.

Application of test item and rinsing:
The plates were incubated for approximately 3 hours at 37 ± 1.0°C. The medium was replaced with fresh DMEM just before the test item was applied. Two tissues were used for a 3-minute exposure and two for a 1-hour exposure. Test item: 50 µl of the undiluted test item was added into the 6-well plates on top of the skin tissues. Negative Control: similarly treated with 50 µl Milli-Q water only. Positive control: 50 µl 8N KOH (potassium hydroxide 8 normal solution). After the exposure periods (3 minutes and 1 hour, respectively), the tissues were washed with phosphate buffered saline to remove residual test item. 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.

All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 59 - 84%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 - 37.0°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 testing laboratory historical data these deviations are not considered significant.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): 8N KOH(aq) (pre-diluted)
Duration of treatment / exposure:
Observations are made 3-minutes and 1-hour post-test item application
Duration of post-treatment incubation (if applicable):
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 (MatTek corporation) over night at room temperature.
Number of replicates:
Duplicate; used for a 3-minute exposure to the test substance and two for a 1-hour exposure.
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 minute exposure
Value:
112
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: mean. Time point: 3 minute exposure. Remarks: n=2 ; CV = 9.8 % ; Score in terms of percentage of negative control.
Irritation / corrosion parameter:
% tissue viability
Remarks:
1 hour exposure
Value:
103
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: mean. Time point: 1 hour exposure. Remarks: n=2 ; CV = 11 % ; Score in terms of percentage of negative control.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data: Negative Control OD570: 3-minutes (n=81): 1.324 – 2.615 ; 1-hour (n=83): 1.361 – 2.352. Positive Control HCD data are presented within the full study report and the concurrent PC was within the specified ranges.

Table 1. Mean absorption and tissue viability in the in vitro skin corrosion test with the test item

 

3-minute application

1-hour application

A (OD570)

B (OD570)

Mean

(OD570)

SD

% viability

CV

A (OD570)

B (OD570)

Mean

(OD570)

SD

% viability

CV

Negative control

1.507

1.531

1.519

±0.017

100

1.5

1.848

1.812

1.830

±0.026

100

2.0

Test item

1.615

1.790

1.730

±0.124

112

9.8

1.767

1.988

1.877

±0.157

103

11.0

Positive control

0.165

0.163

0.164

±0.002

11

1.6

0.144

0.134

0.190

±0.008

7.6

7.5

Values are corrected for background absorption (0.0433). Isopropanol was used to measure the background absorption.

SD = Standard deviation

Duplicate exposures are indicated by A and B.

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Interpretation of results:
GHS criteria not met
Remarks:
EU criteria not met
Conclusions:
Under the conditions of this in vitro study, the test item is not considered to be corrosive to the skin.
Executive summary:

The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test item in accordance with GLP using a human three dimensional epidermal model (EpiDerm (EPI-200)). The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 μl of the undiluted test item was added (with a pipette) into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively. The positive control had a mean relative tissue viability of 7.6% after 1 hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of viability 20 - 100% the Coefficient of Variation between tissue replicates was less than 11%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 112% and 103%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive. Under the conditions of this study, the test item is not corrosive in the in vitro skin corrosion test.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 77%) which does not affect the reliability of the study
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 77%) which does not affect the reliability of the study
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiSkin RHE Small Model (Lot no.: 17-EKIN-044). The test consists of topical application of the test item -one on the skin tissue for 15 minutes. After exposure the skin tissue is thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect is performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The EPISKIN Small 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. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 22 hours at 37°C (Figure 1). Maintenance medium and Assay medium were supplied by a recognised supplier (documented in the full study report).

Application of test item and rinsing:
Test item: Tissues were treated with 10 µl test item for 15 minutes exposure period. Negative control: 10 µl PBS (negative control) was similarly applied. Positive control: 10 µl SDS (5% w/v) was respread after 7 minutes contact time to maintain distribution of the SDS - for the remainder of the contact period. This was not deemed necessary in the negative control or test item definitive test group.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. 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.

All incubations, with the exception of the test item 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 77 - 86%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.0°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 testing laboratory historical data these deviations are not considered significant.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µl
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl
- Concentration (if solution): Not applicable.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl
- Concentration (if solution): 5% SDS (pre-diluted)
Duration of treatment / exposure:
15 ± 0.5 minutes at room temperature, after which the tissues were washed with phosphate buffered saline to remove residual test item.
Duration of post-treatment incubation (if applicable):
Subsequently the skin tissues were incubated for 42 hours at 37°C.
Number of replicates:
Triplicate; treatment and concurrent negative control and positive control groups
Irritation / corrosion parameter:
% tissue viability
Value:
124
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: mean. Time point: 15 minute exposure. Remarks: n=3; SD = 3.5% ; Score in terms of percentage of negative control.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data (n=173): the mean OD of the positive control was 0.13; range 0.023 to 0.437. In this same period the mean OD of the negative control was 0.98; range 0.422 – 1.547.

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test substance

 

A

(OD570)

B

(OD570)

C

(OD570)

Mean

(OD570)

SD

Mean tissue viability (% of control)

Standard Deviation

(%)

Negative control

0.916

0.861

0.934

0.904

±0.038

100

4.2

Test item

1.155

1.114

1.093

1.121

±0.031

124

3.5

Positive control

0.303

0.343

0.340

0.329

±0.022

36

2.4

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

Values are corrected for background adsoption (0.042). Isopropanol was used to measure background adsorption.

Negative control: Phosphate buffered saline (PBS)

Positive control: 5% (aq.) Sodium dodecyl sulphate (SDS)

Interpretation of results:
GHS criteria not met
Remarks:
EU criteria not met
Conclusions:
Under the conditions of this in vitro study, the test item is not considered to be irritating to the skin.
Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. 10 μl of the undiluted test item was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μl PBS (negative control) and 3 tissues with 10 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 item. 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. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 36% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 124%. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment it is considered to be non-irritant. Under the conditions of this study the test item is not irritant in the in vitro skin irritation 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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
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)
Deviations:
no
Qualifier:
equivalent or similar 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)
Remarks:
inspected: September 2015; signature: November 2015
Species:
other: bovine
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse.
- Age at study initiation: not reported
- Weight at study initiation: not reported
- Housing: The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (recognised supplier) 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 1
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 microlitres
- Concentration (if solution): undiluted

Duration of treatment / exposure:
10 ±1 minutes at 32 ± 1ºC.
Duration of post- treatment incubation (in vitro):
After treatment the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed.
Number of animals or in vitro replicates:
Three (3) per test item, or negative or positive controls, respectively.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: 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. Following mounting: the anterior and posterior chambers of each BCOP holder were filled with complete Earle’s Minimum Essential Medium (cMEM) and the holders were incubated at 32 ± 1 ºC for a minimum of 1 hour. After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM.

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically. Only corneas with opacity ≥ 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

NEGATIVE CONTROL USED: physiological saline

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: Ethanol ; > 99.9% purity

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: Yes. Following exposure the holders were incubated, for 120 ± 10 minutes at 32 ± 1°C. A post-treatment opacity reading was taken and each cornea was visually observed.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the
anterior chamber and the cornea was rinsed three times with fresh MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
- POST-EXPOSURE INCUBATION: Following the final opacity measurement the posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg Na-fluorescein/ml cMEM solution Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: 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). A test item that induces an In Vitro Irritancy Score >/=55.1 is defined as an ocular corrosive or severe irritant. A test item with an IVIS
Irritation parameter:
in vitro irritation score
Run / experiment:
mean (n=3)
Value:
0.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No. The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were turbid post treatment and post incubation.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline:
1. Ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the current historical control data (HCD) mean. ACTUAL: PC IVIS range of 34.7 to 78.2. Mean = 48.0.
2. Physiological saline solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values for bovine corneas treated with the respective negative control of the current historical control data (HCD). When testing liquids the negative control limit for opacity should be ≤3.0 and for permeability ≤0.042. ACTUAL: PC IVIS = 1.1, opacity ≤ 1.8 and permeability ≤ 0.007.

Table 1 Summary of opacity, permeability and in vitro scores

Treatment

Mean Opacity #1

Mean Permeability #1

Mean In vitro Irritation Score #1 , 2

Negative Control

1.2

-0.001

1.1

Positive Control

(ethanol)

22

1.778

48.0

Test item

0.1

0.004

0.2

#1 Calculated using the negative control mean opacity and mean permeability values

#2 Mean in vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

 

Table 2 Opacity score

Eye

Opacity before treatment

Opacity after treatment

Final opacity #1

Negative control corrected final opacity #2

Mean Opacity

Negative Control

1

2.9

4.7

1.8

-

1.2

2

4.0

4.4

0.4

-

3

3.1

4.4

1.3

-

Positive Control

4

3.9

30.9

27.0

26

22

5

3.6

23.7

20.1

19

6

1.8

22.8

21.0

20

Test item

7

3.8

5.2

1.4

0.3

0.1

8

2.6

3.2

0.6

0.5

9

3.0

4.8

1.8

0.7

#1 Final Opacity = Opacity after treatment – Opacity before treatment

#2 Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

 

Table 3 Permeability score individual values (uncorrected)

Eye

Dilution Factor

OD490 1

OD490 2

OD490 3

Average OD490

Final OD

Mean Final Negative Control

 

Negative Control

 

1

1

-0.011

-0.004

-0.011

-0.009

-0.009

0.000

2

1

0.002

0.007

0.013

0.007

0.007

3

1

-0.003

-0.004

-0.002

-0.003

-0.003

 

Positive Control

 

4

1

1.320

1.317

1.331

1.323

1.323

5

6

0.332

0.332

0.339

0.334

2.006

6

6

0.339

0.322

0.332

0.331

1.986

 

Test substance

7

1

0.0028

0.013

0.019

0.02

0.020

8

1

-0.007

-0.006

-0.008

-0.007

-0.007

9

1

-0.004

-0.004

-0.007

-0.005

-0.005

 

Table 4 In vitro irritancy score

Eye

Negative control corrected final opacity

Negative control corrected Final OD490

In vitro Irritancy Score #1

Negative Control

1

1.8

-0.009

-0.1

2

0.4

0.007

0.0

3

1.3

-0.003

-0.9

Positive Control

4

26

1.324

46

5

19

2.015

49

6

20

1.995

50

Test substance

7

0.3

0.021

0.6

8

-0.5

-0.006

-0.6

9

0.7

-0.004

0.6

#1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value)

Interpretation of results:
GHS criteria not met
Remarks:
EU criteria not met
Conclusions:
Under the conditions of this in vitro study, the test item is not considered to be irritating to the eye.
Executive summary:

The study was performed to OECD TG 437 and EU Method B.47 to assess the irritancy potential of the test item to the eye following exposure to bovine corneas in accordance with GLP. A total of 3 corneas per treatment group were used. A volume of 750 microlitres of the test item was placed the cornea. The negative control group received physiological saline and the positive control group received neat ethanol. For each group the corneas were incubated for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Earle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. 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 (ethanol) was 48.0 and was within two deviations of the historical positive control data mean and within the historical positive control range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The corneas treated with the test item showed opacity values ranging from -0.5 to 0.7 and permeability values ranging from -0.006 to 0.021 and in vitro irritancy scores ranged from -0.6 to 0.6. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.2 after 10 minutes of treatment. Based on these results the test item is considered to be not irritating or corrosive in the Bovine Corneal Opacity and Permeability test.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Corrosion:

in vivo, OECD TG 431, 2017: The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test item in accordance with GLP using a human three dimensional epidermal model (EpiDerm (EPI-200)). The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 μl of the undiluted test item was added (with a pipette) into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively. The positive control had a mean relative tissue viability of 7.6% after 1 hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of viability 20 - 100% the Coefficient of Variation between tissue replicates was less than 11%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 112% and 103%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive. Under the conditions of this study, the test item is not corrosive in the in vitro skin corrosion test.

 

Skin Irritation:

in vitro, OECD TG 439, 2018: The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. 10 μl of the undiluted test item was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μl PBS (negative control) and 3 tissues with 10 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 item. 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. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 36% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 124%. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment it is considered to be non-irritant. Under the conditions of this study the test item is not irritant in the in vitro skin irritation test.

 

Eye Irritation:

In vitro, OECD TG 437, 2017: The study was performed to OECD TG 437 and EU Method B.47 to assess the irritancy potential of the test item to the eye following exposure to bovine corneas in accordance with GLP. A total of 3 corneas per treatment group were used. A volume of 750 microlitres of the test item was placed the cornea. The negative control group received physiological saline and the positive control group received neat ethanol. For each group the corneas were incubated for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Earle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. 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 (ethanol) was 48.0 and was within two deviations of the historical positive control data mean and within the historical positive control range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The corneas treated with the test item showed opacity values ranging from -0.5 to 0.7 and permeability values ranging from -0.006 to 0.021 and in vitro irritancy scores ranged from -0.6 to 0.6. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.2 after 10 minutes of treatment. Based on these results the test item is considered to be not irritating or corrosive in the Bovine Corneal Opacity and Permeability test.

 

Respiratory Irritation:

No data available.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for dermal irritation.

 

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation.