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Description of key information

The test substance was considered to be irritating to the skin, based on the EpidermTM skin corrosion test and skin irritation test.

The test substance was considered to cause irreversible damage to the eyes, based on a modified Draize test.

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)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): DERIPHAT 160 C
- Physical state:Solid
- Analytical purity: see report AU 122958-1
- Lot/batch No.: 4986V1
- pH-value: 8
- Homogeneity:The test substance was homogeneous by visual inspection
- Stability under test conditions: yes
Test system:
human skin model
Source species:
human
Cell type:
other: cell culture inserts (MILLICELLs, 10 mm)
Cell source:
foreskin from a single donor
Source strain:
other: 24 EpiDerm™ 200 tissues
Vehicle:
water
Details on test system:
EpiDerm™ 200 kit: MatTek Corp., Ashland MA, USA or MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia containing:
24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® 1 cm
Tissue for MTT-reduction control: Epi-200 tissue that is killed by freezing at –20°C

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.
Irritation test:

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, one killed tissue per exposure time was treated with the test substance and NC, respectively, in order to detect direct MTT reduction.

25 µL de-ionized water was applied first. Thereafter, a bulk volume of 25 µL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water.

Control tissues were concurrently applied with 50 µL of de-ionized water (negative control, NC and killed tissue control, KC) or with 50 µL of 8 n potassium hydroxide (positive control, PC) or test substance (killed tissue control, 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 by MTT solution and tissues were incubated for 3 hours.

After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25µL (10mg)
Duration of treatment / exposure:
3 min
Duration of post-treatment incubation (if applicable):
1 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
98
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure
Value:
86
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Conclusion as derived from a WoE in combination with the in vitro irritation test
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)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): DERIPHAT 160 C
- Physical state:Solid
- Analytical purity: see report AU 122958-1
- Lot/batch No.: 4986V1
- pH-value: 8
- Homogeneity: Homogeneous by visual inspection
- Stability under test conditions: yes
Test system:
human skin model
Source species:
human
Cell type:
other: cell culture inserts (MILLICELLs, 10 mm)
Cell source:
foreskin from a single donor
Source strain:
other: 24 EpiDerm™ 200 tissues
Vehicle:
water
Details on test system:
EpiDerm™ 200 kit: MatTek Corp., Ashland MA, USA or MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia containing:
24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® 1 cm
Tissue for MTT-reduction control: Epi-200 tissue that is killed by freezing at –20°C

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.

Different cell culture media are used:
Assay medium: Dulbecco's modified eagle's medium (DMEM); for the assay and for diluting MTT (MatTek Corp., Ashland MA, USA or MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany)
Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca 2+, Mg 2+ (MatTek Corp., Ashland MA, USA or MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Biochrom, Germany)
Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek Corp., Ashland MA, USA or MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma,Germany), 1.0 mg / mL assay medium

Extracting agent: Isopropanol p.a.

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 applied with 30 µL of sterile PBS (negative control, NC) or with 30 µL of 5% SDS (positive control, PC).

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. Subsequently, the tissues were incubated in 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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 µL (10mg)
Duration of treatment / exposure:
1 hour

Duration of post-treatment incubation (if applicable):
42 h at 37°C
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
12
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
modified draize test
Principles of method if other than guideline:
Based on Draize et al. J. Pharmacal. Exp. Ther. 83 : 377-390, 1944.
GLP compliance:
no
Specific details on test material used for the study:
PRODUCT IDENTIFICATION: sodium N-(2-carboxyethyl)-N-dodecyl-β-alaninate, 16% Solids, pH 7.0, #54R-74
PRODUCT DESCRIPTION: Clear yellow liquid.
PSL NO.: E20506-2
PRODUCT IDENTIFICATION: Mirataine H2C-HA
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Three New Zealand White Rabbits from T. Hanna Colony.
- Housing: individually housed in stainless steel wire bottomed cages
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 68 - 72°F.
- Air changes: environmentally controlled room
- Photoperiod (hrs dark / hrs light):12 / 12
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration: 16%

The test material was placed on the everted lower lid of one eye of each rabbit. The upper and lower lids were gently held together for 1 second before releasing, to prevent loss of the test material.
Duration of treatment / exposure:
24h, no washout
Observation period (in vivo):
At 24, 48 and 72 hours, and at 4 and 7 days.
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: no

SCORING SYSTEM: Draize scoring system
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
fully reversible within: 2 days
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3.7
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2.7
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
other: Discharge
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
other: Discharge
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 3 days
Irritation parameter:
other: Discharge
Basis:
animal #3
Time point:
24/48/72 h
Score:
2.7
Max. score:
3
Reversibility:
fully reversible within: 3 days
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2.3
Max. score:
3
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2.3
Max. score:
3
Reversibility:
not fully reversible within: 7 days
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion:

The test item (10% in water) was tested in vivo on rabbits for skin irritation according to the Draize test as described in the Federal Register - Code of the Federal Regulations, Part 191, Chapter I, Title 21, reprinted from Federal Register of August 12, 1961, 26 F.R. 7333. A primary skin irritation score of 0.15 was reported, with no further details given; the substance was described as "a mild irritant" to the skin (General Miles Chemicals Inc, 1963).

A study assessing the dermal corrosion/irritation of the test item by a single topical application of 25 µL bulk volume (about 10 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). Skin irritation was assessed according to the OECD guideline 439 (BASF, 2013) and skin corrosion according to the OECD guideline 431 (BASF, 2013). For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The EpiDerm™ skin corrosion/irritation test showed the following results: The test substance is able to reduce MTT directly. Therefore an additional MTT reduction control was introduced. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing (performed with the corrosion test, only). The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 98%, and it was 86% after an exposure period of 1 hour. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 12%. Based on the observed results it was concluded, that the test item shows a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

 

All available information is taken into account to assess the skin irritating potential of the test substance. The in vitro skin irritation study shows an irritating result based on cell viability. This result is underlined by the in vivo study with 10% A.I. in rabbits. Both test did not show indications for corrosion, therefore the substance should be regarded as a skin irritant.

 

Eye irritation:

A test solution containing 5% dilution of the test item was tested in rabbits for eye irritation according to the Draize test as described in the Federal Register - Code of the Federal Regulations, Part 191, Chapter I, Title 210, reprinted from Federal Register of August 12, 1961, 26 F.R. 7333 (General Mills Chemicals Inc, 1963). The following scoring values were reported for the cornea: 3.0 at 24 h, 0.8 at 48 h and 0 at 72 h. For iris: 5.8 at 24 h, 2.5 at 48 h and 0 at 72 h and conjunctivae (no distinction was made between redness and chemosis): 2.6 at 24 h, 0 at 48 h and 0 at 72 h were reported. The scoring as performed in the study was not in accordance with scoring procedure as currently needed and conducted for purpose of assessment of the eye irritation potential of the test item. Additionally the substance concentration used in the experiment was low. Nevertheless, since scores of 0 were reported at reading time point 72 h for each of cornea, iris and conjunctivae, full reversibility seems to be achieved at that time point.

 

In another study the test substance (16% solids) has been tested for acute ocular irritation in 3 New-Zealand White rabbits according to modified Draize test (PSL, 1982). The test item was instilled in the form of liquid as a single dose of 0.1 mL into the conjunctival sac of one eye of the animal. The other eye remained untreated and served as a control. Ocular examinations were performed at 24, 48, 72 hours, and at day 4, and 7 after treatment. Corneal opacity (grade 2) was observed in 1 out of 3 rabbits at 24-48 hours, and at day 3 to 7 in another (grade 1), over a large area. The mean scores (24-48-72 hours) calculated for each animal were 0.3 – 0.0 – 1.3 for corneal opacity. Iris inflammation was observed in only one animal at 72 hours. Conjunctival redness (grade 2 or 3) was observed at 24-48-72 hours after instillation in all of the animals, and had reversed by day 7 in 1 animal. This effect was scored 1 in the two other animals. Important chemosis was observed in all of the rabbits at 24 hours, and decreased to grade 1 (2/3 animals) or 2 (1/6 animals) by day 4 and 7. The mean scores (24-48-72 hours) calculated for each animal were 2.0 – 2.3 – 2.3 for redness, and 3.7 – 2.7 – 3.0 for chemosis. In addition, ocular discharge was observed in all of the animals throughout the study period. Considering the persistence of ocular damages at the end of the observation period (day 7), the test item is considered to be damaging to the eye.

 

There are two eye irritation studies available conducted not current protocols. Nevertheless the study from PSL (1982) is adequate documented and conducted. As the study also shows the most concerning result this study is used as key study, whereas the other study is only integrate as supporting information.

 

All available information is taken into account to assess the eye irritating potential of the test substance. Based on the general mills (1963) study where as low concentrated test item dilution shows clearly irritating eye effects it is assumed that the neat substance may lead to more detrimental effects. This is confirmed by the key study (PSL, 1982) where the higher concentrated substance leads to non-reversible eye effects. As it is believed that the neat substance has a higher irritating potential the substance is assumed to be severe eye irritating, even as the current test does not cover a 21 d observation period.

Justification for classification or non-classification

Based on the observed skin irritation the test item is classified as Skin Irrit. 2 (H315: Causes skin irritation) in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.

Based on the observed irreversible eye damage the test item is classified as Eye Damage 1 (H318: Causes serious eye damage) in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.