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Description of key information

The test substance shows a skin irritation potential.

The test substance shows an eye irritation potential.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Vehicle:
unchanged (no vehicle)
Details on test system:
Three-dimensional human epidermis model:
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes, which have been cultured to form a multi layered, 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 that found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs, 10 mm ∅) specially prepared and available commercially as kits (EpiDerm™ 200) containing 24 tissues on shipping agarose.
Tissue model: EPI-200
Tissue Lot Number: 28646 (Certificate of Analysis see Appendix)
Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Corrosion test:
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour, but not more than 1.5 hours before test substance application, tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The pre-incubation medium was replaced with fresh medium immediately before application. Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. In addition, two killed control tissues per exposure time were treated with the test substance and the NC, respectively, to detect direct MTT reduction. Fifty microliters (50 μL) undiluted liquid test substance were applied using a pipette. Control tissues were treated concurrently with 50 μL deionized water (NC, NC KC) or with 50 μL 8 N potassium hydroxide solution (PC) or test substance (KC). The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced with MTT solution, and the tissues were incubated for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT incubation. The formazan that was produced metabolically by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes corrosion test
Value:
91
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour corrosion test
Value:
47.4

Exposure period: 3 min

Test substance

 

tissue 1

tissue 2

Mean

SD

CV[%]

NC

mean OD570

2.071

2.098

2.084

 

 

viability

[% of NC]

99.4

100.6

100.0

0.9

0.9

18/0019245-1

mean OD570

2.032

1.761

1.897

 

 

viability

[% of NC]

97.5

84.5

91.0

9.2

10.1

PC

mean OD570

0.167

0.235

0.201

 

 

viability

[% of NC]

8.0

11.3

9.6

2.3

23.8

Exposure period: 1 h

Test substance

 

tissue 1

tissue 2

Mean

SD

CV[%]

NC

mean OD570

2.043

2.084

2.063

 

 

viability

[% of NC]

99.0

101.0

100.0

1.4

1.4

18/0019245-1

mean OD570

1.023

0.932

0.977

 

 

viability

[% of NC]

49.6

45.1

47.4

3.1

6.6

PC

mean OD570

0.122

0.139

0.130

 

 

viability

[% of NC]

5.9

6.7

6.3

0.6

9.5

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results observed and by applying the evaluation criteria, it was concluded that Laromer IPGA shows not a skin corrosion potential in the EpiDerm™ in vitro corrosion test under the test conditions chosen.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Vehicle:
unchanged (no vehicle)
Details on test system:
Three-dimensional human epidermis model
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes, which have been cultured to form a multi layered, 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 that found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs, 10 mm ∅) specially prepared and available commercially as kits (EpiDerm™200) containing 24 tissues on shipping agarose.
Tissue model: EPI-200
Tissue Lot Number: 28646 (Certificate of Analysis see Appendix)
Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Irritation test:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour, the pre-incubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and the NC, respectively. In addition, three killed control tissues were used for the test substance and the NC, respectively, to detect direct MTT reduction. Thirty microliters (30 μL) undiluted liquid test substance were applied using a pipette.
Control tissues were treated concurrently with 30 μL sterile PBS (NC, NC KC) or with 30 μL 5% SDS (PC) or test substance (KC). A nylon mesh was placed carefully onto the tissue surface of the NC, NC KC and PC afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was dried carefully with a sterile cotton swab. Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours, the tissues were transferred into new 6-well plates pre-filled with 0.9 mL fresh medium and placed into the incubator for an additional 18 ± 2-hour post-incubation period.
After the post-incubation period, the assay medium was replaced with 0.3 mL MTT solution, and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT incubation. The formazan that was produced metabolically by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
irritation test
Value:
4

Irritation test:

Test substance

 

tissue 1

tissue 2

tissue 3

mean

SD

NC

mean OD570

2.189

2.172

2.208

2.189

 

viability

[% of NC]

100.0

99.2

100.8

100.0

0.8

18/0245-1

mean OD570

0.080

0.129

0.053

0.087

 

viability

[% of NC]

3.7

5.9

2.4

4.0

1.7

PC

mean OD570

0.047

0.049

0.044

0.056

 

viability

[% of NC]

2.1

2.2

2.0

2.1

0.1

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Based on the results observed and by applying the evaluation criteria, it was concluded that Laromer IPGA shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.
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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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)
GLP compliance:
yes (incl. QA statement)
Details on test animals or tissues and environmental conditions:
Isolated bovine cornea: The test system is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months) Schlachthof Alzey, Germany.

Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32°C for at least 1 hour.
After the equilibration period, the medium in both chambers was replaced by fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 556 opacity units1 were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups. Each corneal holder was uniquely identified with a number on the chambers.

Each treatment group (test substance, the NC and the PC) consisted of 3 corneas. Before application, the medium in the anterior chamber was removed by using a syringe. 750 μL undiluted liquid test substance were applied into the anterior chamber by using a pipette. For the control tissues, 750 μL deionized water (negative control, NC) or 750 μL 100% ethanol / 100% dimethylformamide (positive controls, PC1 / PC2) were applied into the anterior chamber by using a pipette. The corneas were incubated in a horizontal position at about 32°C for approximately 10 minutes (liquids and surfactants). The test substance, the NC and the PC were then removed from the anterior chamber by using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).

The corneas were incubated for 2 further hours at about 32°C. After the incubation period, the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.

Before measurement, each cornea was visually observed and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.

For determination of permeability, the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 minutes in a horizontal position at about 32°C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was spectrophotometrically measured. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

Negative control (NC): Deionized water
Positive control (PC): 100% ethanol (PC1) / 100% dimethylformamide (PC2) for liquid test substances and surfactants
Vehicle:
unchanged (no vehicle)
Controls:
yes
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
6.5

Result BCOP:

Test substance

Mean Opacity Value

Mean Permeability Value

Mean In Vitro Irritancy Score

18/0245-1

6.5

0.002

6.5

NC

4.3

0.001

4.4

PC1

24.8

0.751

36.1

PC2

102.1

0.389

107.9

Summary of individual test results and test strategy evaluation

Test Method

Test Result

Test Evaluation

Evaluation Test Strategy

BCOP Test

The mean IVIS of the corneas treated with the test substance was 6.5.

Not identified as corrosive or severe irritant

 

 

Ocular irritant

EpiOcular

The final relative mean Ocular irritant viability of the tissues treated with the test

substance was 32.5%.

 

Irritant

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria, it was concluded that Laromer IPGA shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes (incl. QA statement)
Details on test animals or tissues and environmental conditions:
EpiOcular:
The EpiOcularTM model (OCL-200) is a three-dimensional, non-keratinized tissue construct composed of normal human-derived, epidermal keratinocytes used to model the human corneal epithelium (compare Figure 1). The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200) containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Tissue Lot Number: 27065 (Certificate of Analysis see Appendix)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Vehicle:
unchanged (no vehicle)
Controls:
yes
Details on study design:
The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results. In order to assess the ability of the test material to reduce MTT directly, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed as described below. The test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently. If the color of the MTT solution or (in case of water-insoluble test substances) the border to the water-phase turned blue / purple, the test substance was presumed to reduce MTT directly. In case of direct MTT reduction, two freeze-killed control tissues (KC) were treated additionally with each the test article and the negative control in the same way. Based on the result of the pretest, it was judged that application of killed control tissues is necessary.

Several test substances were tested in parallel within the present test (test no. 102) by using the same control tissues (NC and PC). Two tissues were treated with each the test substance, the PC and the NC. In addition, two killed tissues were used for each the test substance and the NC to detect direct MTT reduction. There are two separate protocols for liquids and solids differing in exposure time and postincubation period. Due to the physical state of the test substance, the protocol for liquids was applied.

On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour, the pre-incubation medium was replaced by fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.

After pre-incubation, the tissues were pretreated with 20 μL PBS to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

By using a pipette, fifty microliters (50 μL) undiluted liquid test substance were applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL sterile deionized water (NC, NC KC) or with 50 μL methyl acetate (PC) or test substance (KC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.

In order to remove the test substance, the tissues were washed with sterile PBS. For this purpose, the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates pre-filled with 5 mL/well pre-warmed medium (post-soak immersion) to remove residual test substance. After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and
transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 2 hours (post-incubation period).

After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT incubation.
The formazan that was metabolically produced by the tissues was extracted by overnight incubation of the tissues in isopropanol at room temperature or by incubation for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was spectrophotometrically determined. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
Remarks on result:
other: EpiOcular: Mean viability of the test substance was 32.5%.

Test substance

 

tissue 1

tissue 2

mean

inter-tissue variability [%]

NC

mean OD570

2.596

2.598

2.597

 

viability

[% of NC]

100.0

100.0

100.0

0.1

18/0104-1

mean OD570

0.892

0.795

0.844

 

viability

[% of NC]

34.4

30.6

32.5

3.7

PC

mean OD570

0.772

0.769

0.771

 

viability

[% of NC]

29.7

29.6

29.7

0.1

Summary table for turnkey test strategy evaluation:

Test Method

Test Result

Test Evaluation

Evaluation Test Strategy

BCOP Test

The mean IVIS of the corneas treated with the test substance was 6.5.

Not identified as corrosive or severe irritant

 

 

Ocular irritant

EpiOcular

The final relative mean Ocular irritant viability of the tissues treated with the test

substance was 32.5%.

 

Irritant

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria, it was concluded that Laromer IPGA shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion:

The objective was to assess the skin irritation and corrosion potential of Laromer IPGA. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). For the corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. Results of the Corrosion Test (SCT): The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 91.0%, and it was 47.4% after an exposure period of 1 hour. Results of the Irritation Test (SIT): The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 4.0%. Based on the results observed and by applying the evaluation criteria, it was concluded that Laromer IPGA shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Eye irritation:

The objective was to assess the eye irritating potential of Laromer IPGA. By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

BCOP Test: The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL undiluted test substance to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test substance for 10 minutes followed by a 2-hour postincubation period. The mean IVIS of the corneas treated with the test substance was 6.5.

EpiOcular: The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 μL undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™). Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period. The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced. The final relative mean viability of the tissues treated with the test substance was 32.5%. Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria, it was concluded that Laromer IPGA shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Justification for classification or non-classification

Based on the results of the skin and eye irritation testing, the test item is classified as skin irritating cat.2 (H315) and eye irritating cat. 2 (H319) according to Regulation (EC) No 1272/2008 (CLP).