Registration Dossier

Administrative data

Description of key information

Skin irritation: non-corrosive, in vitro EpiDerm OECD TG 431, 2015

Skin irritation: irritating, in vitro EpiSkin OECD TG 431, 2015

Eye irritation: Weight of evidence: irriating, 2019

Eye irritation: non-irritating, in vitro BCOP OECD TG 437, 2015

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:
2015
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: Human Skin Model Test)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 50%) which does not affect the reliability of the study
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 50%) which does not affect the reliability of the study
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: March 2013; signature: May 2013
Species:
other: EpiDerm Skin Model (EPI-200, Lot no.: 21267 kit Q).
Strain:
not specified
Details on test animals and environmental conditions:
EpiDerm Skin Model). The 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.

Freeze-killed tissues. Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 ml DMEM medium. Further use of killed tissues was similar to living tissues.

All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 50 - 87%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.4°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.
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl
- Concentration (if solution): undiluted

Duration of treatment / exposure:
Two tissues were used for a 3-minute exposure to the test substance and two for a 1-hour exposure.
In addition two freeze-killed tissues treated with test substance and two freeze-killed non treated tissues were used for the cytotoxicity evaluation with MTT.
Number of animals:
The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test substance and two for a 1-hour exposure. In addition two freeze-killed tissues treated with test substance and two
freeze-killed non treated tissues were used for the cytotoxicity evaluation with MTT.
Details on study design:
TEST SITE
- Area of exposure: 50 μl of the undiluted test substance was added on top of the skin tissues.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: Two tissues were washed after 3 minutes and two tissues were washed after 1 hour.

SCORING SYSTEM: Skin corrosion is expressed as the remaining cell viability after exposure to the test substance.
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 minutes exposure
Run / experiment:
mean
Value:
87
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Score in terms of percentage of control
Irritation / corrosion parameter:
% tissue viability
Remarks:
1 hour
Run / experiment:
mean
Value:
94
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Scored in terms as percentage of control
Other effects / acceptance of results:
Table 1 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with test substance compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 87% and 94% respectively. The values are corrected for non-specific MTT reaction.

The relative mean tissue viability of the positive control was after the 3-minute and 1-hour treatments compared to the negative control tissues was 8% and 10% respectively.

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

 

3-minute application (OD540)

1-hour application (OD540)

OD540measurement

A

B

Mean

SD

Mean tissue viability (% of control)

A

B

Mean

SD

Mean tissue viability (% of control)

Negative control 

1.651

1.640

1.645

±0.008

100

1.669

1.603

1.636

±0.047

100

Test Substance (1)

1.500

1.362

1.431

±0.098

87

1.549

1.517

1.533

±0.022

94

Positive control 

0.129

0.127

0.128

±0.001

8

0.184

0.153

0.169

±0.022

10

(1) The values are corrected for the non-specific MTT reaction

SD = Standard Deviation

Duplicate Exposures are indicated by A and B

Values are corrected for background absorption (0.043). Isopropanol was used to measure the background absorption.

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the test substance is not considered to be corrosive in the in vitro skin corrosion test using EpiDerm Skin Model.
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 substance 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 substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test substance and two for a 1-hour exposure. The test substance did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Therefore in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test substance and two freeze-killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test substance was 10% of the negative control tissues. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test substance treated viable tissues. Fifty μl of the undiluted test substance 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 8% after 3 minutes exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 17% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 10%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 87% and 94%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test substance is considered to be not corrosive. Under the conditions of this study the test material 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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met. Minor deviation in the use of Freeze-Killed tissues for non-specific direct-MTT reduction determination; which does not affect the reliability of the overall study conclusion.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Minor deviation in the use of Freeze-Killed tissues for non-specific direct-MTT reduction determination; which does not affect the reliability of the study conclusion.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
see above.
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: March 2013; signature: May 2013
Species:
other: EPISKIN Small ModelTM
Strain:
not specified
Details on test animals and environmental conditions:
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.

Killed tissues. Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.

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 82 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.1°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.
Controls:
other: negative and positive controls were used
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µl
- Concentration (if solution): undiluted

Duration of treatment / exposure:
Tissues were treated with the test material for 15 minutes and then washed with phosphate buffered saline to remove residual test material. Subsequently the skin tissues were incubated for 42 hours at 37°C.
In addition three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT.
Details on study design:
TEST SITE
- Area of exposure: 10 μl of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 15 minutes

SCORING SYSTEM: Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Irritation / corrosion parameter:
% tissue viability
Remarks:
15 ± 0.5 minutes exposure
Run / experiment:
mean
Value:
41
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minute exposure. Reversibility: no data. Remarks: Score in terms of percentage of negative control.

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

  OD570 Mean  SD Mean tissue viability (% of control)
A B C
Negative control 1.099 0.964 1.188 1.084 ±0.113 100
Test Substance (1) 0.235 0.598 0.488 0.441 ±0.186 41
Positive control 0.071 0.107 0.113 0.097 ±0.023 9

OD = optical density SD = Standard deviation Triplicate exposures are indicated by A, B and C.

Test Substance (1) Values are corrected for the non-specific MTT reaction.

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:
Category 2 (irritant) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this in vitro study, the test material is considered to be irritating to 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 substance 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 substance together with negative and positive controls. Ten μl of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues. The test substance did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test substance was 17% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable 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 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. 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 9% after 15 ± 0.5 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 standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 41%. Since the mean relative tissue viability for the test substance was below 50% after 15 minutes treatment it is considered to be irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
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 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
Qualifier:
according to
Guideline:
other: The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM)
Deviations:
no
Qualifier:
according to
Guideline:
other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
Deviations:
no
Qualifier:
according to
Guideline:
other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: March 2013; signature: May 2013
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:
other: Three corneas were used for the positive and negative control groups.
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
Observation period (in vivo):
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 corneas were used for each treatment group
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): After treatment with the test material the epithelium was washed with MEM with phenol red (Eagle’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.
- Time after start of exposure: 10 ± 1 minutes

SCORING SYSTEM:
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. A test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant. A IVIS score of less than 3 is interpreted as having no prediction for corrosive or irritant effects.

TOOL USED TO ASSESS SCORE: Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2)
Irritation parameter:
in vitro irritation score
Remarks:
10 minutes exposure
Run / experiment:
mean
Value:
1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
IVIS scores ranged from -0.1 to 2 after 10 minutes treatment
Other effects / acceptance of results:
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 (10% (w/v) Benzalkonium Chloride) was 128.2 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.3 after 10 minutes of treatment. Additionally, all opacity and permeability responses were within the ranges of the historic negative controls.

No pH effect of the test substance was observed on the rinsing medium.

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

-0.3

0.000

0.0

Positive Control

(Benzalkonium chloride)

81.7

3.102

128.2

Test substance

1.3

-0.004

1.3

#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

1

1

0

0.0

0.0

2

0

0

0

0.0

3

0

0

0.0

0.0

Positive Control

4

-1

83

84

84.0

81.7

5

-1

83

84

84.0

6

-1

76

77

77.0

Test substance

7

-2

0

2

2.0

1.3

8

0

0

0

0.0

9

0

2

2

2.0

#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

0.010

0.011

0.011

0.011

0.011

0.008

2

1

0.004

0.004

0.004

0.004

0.004

3

1

0.009

0.007

0.008

0.003

0.008

 

Positive Control

 

4

6

0.314

0.311

0.312

0.312

1.874

5

6

0.482

0.479

0.473

0.478

2.868

6

6

0.794

0.781

0.775

0.783

4.700

 

Test substance

7

1

0.003

0.002

0.003

0.003

0.003

8

1

0.001

0.002

0.001

0.001

0.001

9

1

0.010

0.006

0.008

0.008

0.008

 

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

0.0

0.003

0.0

2

0.0

-0.004

-0.1

3

0.0

0.000

0.0

Positive Control

4

84.0

1.829

111.4

5

84.0

2.823

126.3

6

77.0

4.655

146.8

Test substance

7

2.0

-0.005

1.9

8

0.0

-0.006

-0.1

9

2.0

0.000

2.0

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

Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions the test material is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test.
Executive summary:

The study was performed to OECD TG 437 and EU Method B.47 to assess the irritancy potential of the test material 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 material was placed the cornea. The negative control group received saline and the positive control group received 10% benzalkonium chloride. 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 (Eagle’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 (10% (w/v) Benzalkonium Chloride) was 128.2 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.3 after 10 minutes of treatment. All opacity and permeability responses were within the ranges of the historic negative controls. Based on these results the test material is considered to be not irritating or corrosive in the Bovine Corneal Opacity and Permeability test.

Endpoint:
eye irritation: in vitro / ex vivo
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Regulation (EC) No. 1907/2006 Annex VII, column 2 section 8.2 the in vitro for eye irritation needs to be conducted in the absence of existing data. This does not need to be conducted if the available information indicates that the criteria are met for classification as corrosive to the skin or irritating to eyes. Within Step 3 and in accordance with ECHA Guidance R.7.a: and following the testing and assessment strategy in section R.7.2.11 – a top down approach was adopted by the applicant and a new BCOP assay has been completed. Due to MTT-reaction (demonstrated in skin irritation RHE in vitro assays) it is unclear if the test substance is fully within the domain of the EPIOCULAR EIT, OECD TG 492 (2015) assay due to the potential for the test item to cause non-specific MTT reduction. In that assay if NSMTT > 30% relative to a negative control is observed there is the potential for the assay to be confounded. On this basis, and in accordance with the tonnage driven information requirements of REACH Regulation (EC) 1907/2006 as amended; the applicant adapts any further information requirement within this annex and applies a precautionary Eye Irritation Category 2 classification, until additional information becomes available and contributes to the weight of evidence for removal or continued application of the classification.
Endpoint conclusion
Endpoint conclusion:
no study available

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Key data : In vitro, OECD TG 439, 2015 : The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test substance 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 substance together with negative and positive controls. Ten μl of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues. The test substance did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test substance was 17% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable 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 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. 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 9% after 15 ± 0.5 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 standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 41%. Since the mean relative tissue viability for the test substance was below 50% after 15 minutes treatment it is considered to be irritant.

 

Skin corrosion:

Key data : In vitro, OECD TG 431, 2015 : The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test substance 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 substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test substance and two for a 1-hour exposure. The test substance did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Therefore in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test substance and two freeze-killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test substance was 10% of the negative control tissues. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test substance treated viable tissues. Fifty μl of the undiluted test substance 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 8% after 3 minutes exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 17% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 10%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 87% and 94%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test substance is considered to be not corrosive. Under the conditions of this study the test material is not corrosive in the in vitro skin corrosion test.

 

Eye irritation/corrosion:

Key data : In vitro, OECD TG 437, 2015 : The study was performed to OECD TG 437 and EU Method B.47 to assess the irritancy potential of the test material 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 material was placed the cornea. The negative control group received saline and the positive control group received 10% benzalkonium chloride. 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 (Eagle’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 (10% (w/v) Benzalkonium Chloride) was 128.2 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.3 after 10 minutes of treatment. All opacity and permeability responses were within the ranges of the historic negative controls. Based on these results the test material is considered to be not irritating or corrosive in the Bovine Corneal Opacity and Permeability test.

 

The applicant has on the basis of the weight of evidence concluded a precautionary Eye Irritation Category 2 classification.

Justification for classification or non-classification

The substance does meet classification criteria under Regulation (EC) No 1272/2008 for skin irritation category 2: H315

The substance does meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation category 2: H319

For skin irritation, the weight of evidence indicates that the substance has the potential to be irritating and sufficient for classification purposes. Available data indicates negative skin corrosion in an in vitro skin corrosion study. An in vitro skin irritation study subsequently indicated that the substance is predicted to be irritating to human skin. The overall evidence by expert judgement indicates that the substance may produce irritation by the dermal route which is sufficient for classification in accordance with the above criteria.

 

For eye irritation, the weight of evidence assessment can be summarised as follows:

1. An available in vitro bovine opacity and corneal permeability (BCOP) assay. Specifically the BCOP IVIS score was < 3.0 indicates that the substance has the potential to cause transient mild irritating effects to the eye but which are insufficient for classification. 2. There is however, discordance between skin and eye irritation conclusions and/or by expert judgement an indication of a belief that within the weight of evidence without additional information there is presently inadequate to conclude negative eye irritation potential.

2. In accordance with ECHA Guidance R.7.a: and following the testing and assessment strategy in section R.7.2.11 – a top down approach was adopted by the applicant and a new BCOP assay has been completed. Due to MTT-reaction (demonstrated in skin irritation RHE in vitro assays) it is unclear if the test substance is fully within the domain of the EPIOCULAR EIT, OECD TG 492 (2015) assay due to the potential for the test item to cause non-specific MTT reduction. In that assay if NSMTT > 30% relative to a negative control is observed there is the potential for the assay to be confounded. On this basis, and in accordance with the tonnage driven information requirements of REACH Regulation (EC) 1907/2006 as amended; the applicant applies a precautionary Eye Irritation Category 2 classification, until additional information becomes available and contributes to the weight of evidence for removal or continued application of the classification. Specifically, at the next tonnage level; if information is generated in accordance with this regulation.

 

References:

1. ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.2.11, October 2015)

2. Guidance on Application of the CLP Criteria, ECHA, version 4.1, June 2015