Registration Dossier

Administrative data

Description of key information

No irritating potential to skin or eyes was detected in three in vitro test systems.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD Guideline 439 and GLP.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Species:
other: 3D human epidermis model (EpiDermTM)
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
25 μL bulk volume (about 6 mg)
Duration of treatment / exposure:
1 h
Observation period:
42 h
Details on study design:
TEST SYSTEM:
The present test is based on the experience that corrosive and irritant chemicals produce cytotoxicity in human reconstructed epidermis after a short term topical exposure. The test is designed to predict a skin corrosion or irritation potential of a chemical by using the three dimensional human epidermis model EpiDermTM. After application of the test material to the stratum corneum surface of the EpiDermTM tissue the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol-extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. Optical density of the extracts of test-substance treated tissues is compared to negative control values from tissues and expressed as relative tissue viability.
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 those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs(R), 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

DIRECT MTT REDUCTION:
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. To assess the ability of the test material to directly reduce MTT a pretest was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested
concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. In case that direct MTT reduction occurred, one freeze-killed control tissue per exposure time was treated with, each, the test article and the negative control.

BASIC PROCEDURE:
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 preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
Three tissues were treated with the test substance, the PC and NC, respectively. 25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid. Control tissues were concurrently treated with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface 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 carefully dried 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 of fresh medium and placed into the incubator for additional 18 ± 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 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 6 microtiter wells filled with isopropanol for each microtiter plate.
Irritation / corrosion parameter:
other:
Value:
95
Remarks on result:
other:
Remarks:
Basis: mean. Max. score: 100.0. (migrated information)

Table 1: Individual and mean OD570 values, individual and mean viability values and standard deviations.

Test

substance

 

tissue 1

tissue 2

tissue 3

mean           SD

 

NC

mean OD570

2.062

2.194

2.048

2.102

viability

[% of NC]

98.1

104.4

97.5

100

3.82

 

NM01

mean OD570

2.208

1.757

2.012

1.993

viability

[% of NC]

105.1

83.6

95.8

95

10.76

 

PC

mean OD570

0.048

0.050

0.057

0.052

viability

[% of NC]

2.3

2.4

2.7

2

0.23

Historical control data of NC and PC of irritation test (Period: Jan 2012 - May 2014).

Historic Range of NC: OD570 = 2.312, SD = 0.248

Historic Range of PC: OD570 = 0.0083, SD = 0.052

Viability (%): Mean = 3.5%, SD = 2.53%

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the observed results and applying the evaluation criteria it was concluded, that NM01 does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.
Executive summary:

The potential of NM01 to cause dermal irritation was assessed by a single topical application of 25 μL bulk volume (about 6 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). Three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a

tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The test substance was not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 95%. It was therefore concluded that NM01 does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
EpiOcular(TM) test
Reference:
Composition 1
Qualifier:
no guideline followed
Version / remarks:
There are no official national or international guidelines for the EpiOcularTM test yet; however,
the test was performed according to the methods described in the following publications:
• MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct:
Procedure details, Version 3.1a of February 10, 2010.
Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye
Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter
Laboratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of
Toxicology, March 2009.
Principles of method if other than guideline:
The test is based on the experience that irritant chemicals produce cytotoxicity in human
reconstructed cornea after a short term topical exposure. The test is designed to predict an
eye irritation potential of a chemical by using the three dimensional human cornea model
EpiOcularTM. After application of the test material to the surface of the EpiOcularTM tissue the
induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is
expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial
dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanolextraction
of the formazan from the tissues, the optical density of the extract is determined
spectrophotometrically. Optical density of the extracts of test-substance treated tissues is
compared to values from negative control tissues and expressed as relative tissue viability.
GLP compliance:
yes
Test material information:
Composition 1
Species:
other: human reconstructed cornea in vitro model
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
50 μL
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
To assess the ability of the test material to directly reduce MTT a pretest was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
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 case where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in thefollowing section.

Basic procedure
Several test substances were tested in parallel within the present test (test no. 50) using the same control tissues (NC and PC). Two tissues were treated with each, the test substance, the PC and the NC. There are two separate protocols for liquids and solids, differing in exposure time and postincubation period. Due to the physical condition of the test substance the protocol for solids was applied.

Pre-incubation of the tissues
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 preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.
Pretreatment of the tissues
After the pre-incubation, the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
Application of the test substance
Using a sharp spoon, a bulk volume of 50 μL of the test material was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.

Removal of the test substance and postincubation period
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 prewarmed medium (post-soak immersion) in order to remove residual test substance. After 25 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 18 hours (postincubation period).

MTT incubation
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 incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. 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.

Data evaluation
The OD570 values determined for the various tissues are measures of their viability. The ratio of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material was an irritant.
Calculation of individual and mean optical densities
The corrected measured OD570 value for each individual tissue was calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way was calculated.
Tissue viability
The quantification of tissue viability is presented as the ratio of the mean OD570 divided by the respective OD570 NC value in percent.

Decision criteria of EpiOcular.
Mean tissue viability (% of negative control)
≤ 60: irritant
> 60: non-irritant
Irritation parameter:
other: tissue viability in % of negative control based on fluorescein leakage
Value:
110
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Interpretation of results:
GHS criteria not met
Executive summary:

The eye irritating potential of the test substance was determined in the in vitro EpiOcular Eye Irritation Test.

The potential of NM01 to cause ocular irritation was assessed by a single topical application of 50 μL bulk volume (about 9 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™).

Two EpiOcular™ tissue samples were incubated with the test substance for 6 hours followed by a 18-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

The EpiOcular™ eye irritation test showed the following results:

The test substance is not able to reduce MTT directly.

The mean viability of the test-substance treated tissues was 110%.

Based on the observed results for the EpiOcular Test alone and applying the evaluation criteria it was concluded, that NM01 does not show an eye irritation potential under the test conditions chosen.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

As no irritating potential to skin or eyes was detected in three established in vitro test systems, the classification criteria according to the CLP Regulation are not met.